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Search results for the GEO ID: GSE47676

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GSM1154495

GPL1355

MCL-Injured-Day 3-Rep 1

MCL-injured_collected at day 3 post-injury

strain: Wistar gender: male weight: 275-299g tissue: medial collateral ligament (MCL) sample group: day 3 post-injury

SAMPLE 1

Sample_geo_accession	GSM1154495
Sample_status	Public on Jun 06 2013
Sample_submission_date	Jun 05 2013
Sample_last_update_date	Jun 06 2013
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Rattus norvegicus
Sample_taxid_ch1	10116
Sample_treatment_protocol_ch1	Animals were subjected to bilateral MCL transections. At day 3 (n=9) or 7 post-injury (n=9), tissue was collected and used for microarray. Another 9 rats were used for intact MCL collection which served as the intact control.
Sample_growth_protocol_ch1	Ligaments were snap frozen in liquid nitrogen immediately upon removal from the body. Tissue was stored at -70C until use
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was isolated from the MCLs combining the TRIzol (Invitrogen, Carlsbad, CA) method with column fractionalization steps of the RNeasy Total RNA kit (Qiagen, Valencia, CA). Tissue was homogenized using a PowerGen 500 homogenizer (Fisher Scientific, Pittsburgh, PA). Subsequent to RNA isolation, yield and purity of RNA was quantified by nanodrop spectrophotometric measurement at 260 nm absorbance (Nanodrop Technologies, Wilmington, DE). Total RNA was converted to double-stranded cDNA using the oligo dT primer containing the T7 RNA polymerase promoter (5’ – GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-T24-3’).
Sample_label_ch1	Biotin
Sample_label_protocol_ch1	Second strand synthesis was performed by incubation with 1x second strand buffer, 200 μM dNTPs, 10 U DNA Ligase (Invitrogen, Carlsbad, CA), 40 U DNA Polymerase I (Invitrogen, Carlsbad, CA), and 2 U RNaseH (Invitrogen, Carlsbad, CA) for two hours at 16°C followed by a five minute incubation with 10 U T4 Polymerase (Invitrogen, Carlsbad, CA). Double stranded cDNA was purified via GeneChip Sample Cleanup Module kit (Affymetrix, Santa Clara, CA Inc.). The biotin-labeled in vitro transcription reaction was performed using the cDNA template and the Affymetrix 3’-Amplification IVT Labeling kit. Briefly, double stranded cDNA was incubated with 1x IVT Labeling buffer, 1x Labeling NTP mix, and 1x T7 Labeling Enzyme mix for 16 hours (37°C) to yield cRNA. The cRNA was purified using the GeneChip Sample Cleanup Module. cRNA yield was determined by absorbance of 260 nM. The cRNA was fragmented to a 0.5 µg/µl final concentration in 1x fragmentation buffer (40 mM Tris-acetate, pH 8.1, 100 mM potassium acetate, and 30 mM magnesium acetate) for 32 minutes at 94°C. The sizeof the cRNA before (0.1 kb and longer) and after (35-200 base fragments) was checked by agarose gel electrophoresis.
Sample_hyb_protocol	GeneChips® (Affymetrix, Santa Clara, CA) were hybridized with biotin labeled cRNA in 300 µl, in 1 X Hybridization buffer (100 mM MES, 1M NaCl (Ambion, Austin, TX), 20 mM EDTA (Ambion, Austin, TX), and 0.01% Tween-20 (Pierce Chemical)), 50 pM control oligonucleotide B2 (Affymetrix, Santa Clara, CA), 0.1 mg/ml Herring Sperm DNA (Promega, Madison, WI Corporation), 0.5 mg/ml acetylated BSA (Invitrogen, Carlsbad, CA) and 1 X Eukaryotic Hybridization Controls (bioB, bioC, bioD, and cre at 1.5, 5, 25 and 100 pM respectively) (Affymetrix, Santa Clara, CA) for 16 hours at 45°C on a rotisserie at 60 rpm. Prior to application to the GeneChip®, samples were heated (95°C for 5 minutes), incubated (45°C for five minutes), and spun (14,000-x g for five minutes). Following hybridization, the labeled samples were removed from the GeneChip®, stored in the appropriate vial at –20C, and filled with non-stringent buffer A containing 6X SSPE (Ambion, Austin, TX), 0.01% Tween 20. All GeneChips® were post processed using the automated Affymetrix GeneChip® Fluidics Station 450. The post processing protocol of the Rat 2.0 Genome GeneChip® included: one wash (10 cycles with 2 mixes/cycle) of non-stringent buffer A at 25°C; one wash of 4 cycles comprised of 15 mixes/cycle with stringent buffer B (100mM MES, 0.1 M NaCl, 0.01% Tween 20) at 50C; staining of the probe array for 10 minutes at 25C in Streptavidin-Phycoerythrin (SAPE) solution (1X MES stain buffer (100 mM MES, 1 M NaCl, 0.05% Tween 20), 2 mg/ml acetylated BSA, 10 µg/ml SAPE (Molecular Probes); post staining consisting of 10 wash cycles of 4 mixes/cycle with non-stringent buffer A at 25°C; a second staining of the probe array for 10 minutes in antibody solution (1X MES stain buffer, 2 mg/ml acetylated BSA, 0.1 mg/ml Normal Goat IgG (Sigma-Aldrich, St. Louis, MO), 3 µg/ml biotinylated antibody (Vector Laboratories); a third stain of the probe array for 10 minutes in SAPE solution at 25C; and a final wash- 15 cycles of 4 mixes/cycle with non-stringent buffer A at 30°C.
Sample_scan_protocol	To quantify flourescence from each feature on the GeneChip, all GeneChips were scanned at a wavelength of 570 nm, using the GC3000 G7 scanner. Flourescent signals corresponding to hybridization intensities were analyzed via Affymetrix GCOS software
Sample_data_processing	Quantification of GeneChips was performed by Affymetrix GeneChip Operating Software (AGCC version 2.0).
Sample_data_processing	The data were compiled with EzArray using Affymetrix default analysis settings and global scaling as normalization method. Data were analyzed using the two-sample Welch t-test to determine significance per gene. To minimize the false discovery rate, the Storey q-value method was performed and significance was based on q < .01. Any Q-value less than or equal to 0.01 per gene was considered significant. Paired t-tests were used to evaluate differences between intact and day 3, intact and day 7, or day 3 and day 7. A critical value of .05 was considered as the criterion to select a significant fold change in gene expression between days. Additionally, microarray data were log transformed [log (base 2)] and the log ratio between the intact ligament with day 3 or day 7 was calculated and expressed as fold change (e.g. (mean day 3-mean intact control)= day 3 difference; fold change=2^(day 3 difference). A gene was considered up or down regulated if the day 3 or day 7 samples were at least 2 fold different than the intact controls.Data are presented as fold change of the means of day 3 and day 7 from the intact ligament values (fold change ± S.D.). Microarray data were functionally grouped using the Database for Annotation, Visualization, and Integrated Discovery (D.A.V.I.D.; http://david.abcc.ncifcrf.gov), integrating functional genomic annotations with intuitive graphical summaries. Using the Affymetrix probes as identifiers, the samples were subjected to functional annotation clustering based on function and gene ontology (GO). The categories of interest were further based on the enrichment score (ES) of the gene group. The ES, determined by the minus log transformation of the geometric mean p-values from all enriched annotation terms, ranks the biological significance of a gene cluster based on the scores of all the annotation terms.
Sample_platform_id	GPL1355
Sample_contact_name	Ray,,Vanderby
Sample_contact_laboratory	WIMR Room 5068
Sample_contact_department	Orthopedics and Rehabilitation
Sample_contact_institute	University of Wisconsin
Sample_contact_address	1111 Highland Ave
Sample_contact_city	Madison
Sample_contact_state	WI
Sample_contact_zip/postal_code	53705
Sample_contact_country	USA
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1154nnn/GSM1154495/suppl/GSM1154495_d3_1_3_left_RG230_2.0_110707.CEL.gz
Sample_series_id	GSE47676
Sample_data_row_count	31099

GSM1154496

GPL1355

MCL-Injured-Day 3-Rep 2

MCL-injured_collected at day 3 post-injury

strain: Wistar gender: male weight: 275-299g tissue: medial collateral ligament (MCL) sample group: day 3 post-injury

SAMPLE 2

Sample_geo_accession	GSM1154496
Sample_status	Public on Jun 06 2013
Sample_submission_date	Jun 05 2013
Sample_last_update_date	Jun 06 2013
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Rattus norvegicus
Sample_taxid_ch1	10116
Sample_treatment_protocol_ch1	Animals were subjected to bilateral MCL transections. At day 3 (n=9) or 7 post-injury (n=9), tissue was collected and used for microarray. Another 9 rats were used for intact MCL collection which served as the intact control.
Sample_growth_protocol_ch1	Ligaments were snap frozen in liquid nitrogen immediately upon removal from the body. Tissue was stored at -70C until use
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was isolated from the MCLs combining the TRIzol (Invitrogen, Carlsbad, CA) method with column fractionalization steps of the RNeasy Total RNA kit (Qiagen, Valencia, CA). Tissue was homogenized using a PowerGen 500 homogenizer (Fisher Scientific, Pittsburgh, PA). Subsequent to RNA isolation, yield and purity of RNA was quantified by nanodrop spectrophotometric measurement at 260 nm absorbance (Nanodrop Technologies, Wilmington, DE). Total RNA was converted to double-stranded cDNA using the oligo dT primer containing the T7 RNA polymerase promoter (5’ – GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-T24-3’).
Sample_label_ch1	Biotin
Sample_label_protocol_ch1	Second strand synthesis was performed by incubation with 1x second strand buffer, 200 μM dNTPs, 10 U DNA Ligase (Invitrogen, Carlsbad, CA), 40 U DNA Polymerase I (Invitrogen, Carlsbad, CA), and 2 U RNaseH (Invitrogen, Carlsbad, CA) for two hours at 16°C followed by a five minute incubation with 10 U T4 Polymerase (Invitrogen, Carlsbad, CA). Double stranded cDNA was purified via GeneChip Sample Cleanup Module kit (Affymetrix, Santa Clara, CA Inc.). The biotin-labeled in vitro transcription reaction was performed using the cDNA template and the Affymetrix 3’-Amplification IVT Labeling kit. Briefly, double stranded cDNA was incubated with 1x IVT Labeling buffer, 1x Labeling NTP mix, and 1x T7 Labeling Enzyme mix for 16 hours (37°C) to yield cRNA. The cRNA was purified using the GeneChip Sample Cleanup Module. cRNA yield was determined by absorbance of 260 nM. The cRNA was fragmented to a 0.5 µg/µl final concentration in 1x fragmentation buffer (40 mM Tris-acetate, pH 8.1, 100 mM potassium acetate, and 30 mM magnesium acetate) for 32 minutes at 94°C. The sizeof the cRNA before (0.1 kb and longer) and after (35-200 base fragments) was checked by agarose gel electrophoresis.
Sample_hyb_protocol	GeneChips® (Affymetrix, Santa Clara, CA) were hybridized with biotin labeled cRNA in 300 µl, in 1 X Hybridization buffer (100 mM MES, 1M NaCl (Ambion, Austin, TX), 20 mM EDTA (Ambion, Austin, TX), and 0.01% Tween-20 (Pierce Chemical)), 50 pM control oligonucleotide B2 (Affymetrix, Santa Clara, CA), 0.1 mg/ml Herring Sperm DNA (Promega, Madison, WI Corporation), 0.5 mg/ml acetylated BSA (Invitrogen, Carlsbad, CA) and 1 X Eukaryotic Hybridization Controls (bioB, bioC, bioD, and cre at 1.5, 5, 25 and 100 pM respectively) (Affymetrix, Santa Clara, CA) for 16 hours at 45°C on a rotisserie at 60 rpm. Prior to application to the GeneChip®, samples were heated (95°C for 5 minutes), incubated (45°C for five minutes), and spun (14,000-x g for five minutes). Following hybridization, the labeled samples were removed from the GeneChip®, stored in the appropriate vial at –20C, and filled with non-stringent buffer A containing 6X SSPE (Ambion, Austin, TX), 0.01% Tween 20. All GeneChips® were post processed using the automated Affymetrix GeneChip® Fluidics Station 450. The post processing protocol of the Rat 2.0 Genome GeneChip® included: one wash (10 cycles with 2 mixes/cycle) of non-stringent buffer A at 25°C; one wash of 4 cycles comprised of 15 mixes/cycle with stringent buffer B (100mM MES, 0.1 M NaCl, 0.01% Tween 20) at 50C; staining of the probe array for 10 minutes at 25C in Streptavidin-Phycoerythrin (SAPE) solution (1X MES stain buffer (100 mM MES, 1 M NaCl, 0.05% Tween 20), 2 mg/ml acetylated BSA, 10 µg/ml SAPE (Molecular Probes); post staining consisting of 10 wash cycles of 4 mixes/cycle with non-stringent buffer A at 25°C; a second staining of the probe array for 10 minutes in antibody solution (1X MES stain buffer, 2 mg/ml acetylated BSA, 0.1 mg/ml Normal Goat IgG (Sigma-Aldrich, St. Louis, MO), 3 µg/ml biotinylated antibody (Vector Laboratories); a third stain of the probe array for 10 minutes in SAPE solution at 25C; and a final wash- 15 cycles of 4 mixes/cycle with non-stringent buffer A at 30°C.
Sample_scan_protocol	To quantify flourescence from each feature on the GeneChip, all GeneChips were scanned at a wavelength of 570 nm, using the GC3000 G7 scanner. Flourescent signals corresponding to hybridization intensities were analyzed via Affymetrix GCOS software
Sample_data_processing	Quantification of GeneChips was performed by Affymetrix GeneChip Operating Software (AGCC version 2.0).
Sample_data_processing	The data were compiled with EzArray using Affymetrix default analysis settings and global scaling as normalization method. Data were analyzed using the two-sample Welch t-test to determine significance per gene. To minimize the false discovery rate, the Storey q-value method was performed and significance was based on q < .01. Any Q-value less than or equal to 0.01 per gene was considered significant. Paired t-tests were used to evaluate differences between intact and day 3, intact and day 7, or day 3 and day 7. A critical value of .05 was considered as the criterion to select a significant fold change in gene expression between days. Additionally, microarray data were log transformed [log (base 2)] and the log ratio between the intact ligament with day 3 or day 7 was calculated and expressed as fold change (e.g. (mean day 3-mean intact control)= day 3 difference; fold change=2^(day 3 difference). A gene was considered up or down regulated if the day 3 or day 7 samples were at least 2 fold different than the intact controls.Data are presented as fold change of the means of day 3 and day 7 from the intact ligament values (fold change ± S.D.). Microarray data were functionally grouped using the Database for Annotation, Visualization, and Integrated Discovery (D.A.V.I.D.; http://david.abcc.ncifcrf.gov), integrating functional genomic annotations with intuitive graphical summaries. Using the Affymetrix probes as identifiers, the samples were subjected to functional annotation clustering based on function and gene ontology (GO). The categories of interest were further based on the enrichment score (ES) of the gene group. The ES, determined by the minus log transformation of the geometric mean p-values from all enriched annotation terms, ranks the biological significance of a gene cluster based on the scores of all the annotation terms.
Sample_platform_id	GPL1355
Sample_contact_name	Ray,,Vanderby
Sample_contact_laboratory	WIMR Room 5068
Sample_contact_department	Orthopedics and Rehabilitation
Sample_contact_institute	University of Wisconsin
Sample_contact_address	1111 Highland Ave
Sample_contact_city	Madison
Sample_contact_state	WI
Sample_contact_zip/postal_code	53705
Sample_contact_country	USA
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1154nnn/GSM1154496/suppl/GSM1154496_d3_4_6_left_RG230_2.0_110707.CEL.gz
Sample_series_id	GSE47676
Sample_data_row_count	31099

GSM1154497

GPL1355

MCL-Injured-Day 3-Rep 3

MCL-injured_collected at day 3 post-injury

strain: Wistar gender: male weight: 275-299g tissue: medial collateral ligament (MCL) sample group: day 3 post-injury

SAMPLE 3

Sample_geo_accession	GSM1154497
Sample_status	Public on Jun 06 2013
Sample_submission_date	Jun 05 2013
Sample_last_update_date	Jun 06 2013
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Rattus norvegicus
Sample_taxid_ch1	10116
Sample_treatment_protocol_ch1	Animals were subjected to bilateral MCL transections. At day 3 (n=9) or 7 post-injury (n=9), tissue was collected and used for microarray. Another 9 rats were used for intact MCL collection which served as the intact control.
Sample_growth_protocol_ch1	Ligaments were snap frozen in liquid nitrogen immediately upon removal from the body. Tissue was stored at -70C until use
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was isolated from the MCLs combining the TRIzol (Invitrogen, Carlsbad, CA) method with column fractionalization steps of the RNeasy Total RNA kit (Qiagen, Valencia, CA). Tissue was homogenized using a PowerGen 500 homogenizer (Fisher Scientific, Pittsburgh, PA). Subsequent to RNA isolation, yield and purity of RNA was quantified by nanodrop spectrophotometric measurement at 260 nm absorbance (Nanodrop Technologies, Wilmington, DE). Total RNA was converted to double-stranded cDNA using the oligo dT primer containing the T7 RNA polymerase promoter (5’ – GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-T24-3’).
Sample_label_ch1	Biotin
Sample_label_protocol_ch1	Second strand synthesis was performed by incubation with 1x second strand buffer, 200 μM dNTPs, 10 U DNA Ligase (Invitrogen, Carlsbad, CA), 40 U DNA Polymerase I (Invitrogen, Carlsbad, CA), and 2 U RNaseH (Invitrogen, Carlsbad, CA) for two hours at 16°C followed by a five minute incubation with 10 U T4 Polymerase (Invitrogen, Carlsbad, CA). Double stranded cDNA was purified via GeneChip Sample Cleanup Module kit (Affymetrix, Santa Clara, CA Inc.). The biotin-labeled in vitro transcription reaction was performed using the cDNA template and the Affymetrix 3’-Amplification IVT Labeling kit. Briefly, double stranded cDNA was incubated with 1x IVT Labeling buffer, 1x Labeling NTP mix, and 1x T7 Labeling Enzyme mix for 16 hours (37°C) to yield cRNA. The cRNA was purified using the GeneChip Sample Cleanup Module. cRNA yield was determined by absorbance of 260 nM. The cRNA was fragmented to a 0.5 µg/µl final concentration in 1x fragmentation buffer (40 mM Tris-acetate, pH 8.1, 100 mM potassium acetate, and 30 mM magnesium acetate) for 32 minutes at 94°C. The sizeof the cRNA before (0.1 kb and longer) and after (35-200 base fragments) was checked by agarose gel electrophoresis.
Sample_hyb_protocol	GeneChips® (Affymetrix, Santa Clara, CA) were hybridized with biotin labeled cRNA in 300 µl, in 1 X Hybridization buffer (100 mM MES, 1M NaCl (Ambion, Austin, TX), 20 mM EDTA (Ambion, Austin, TX), and 0.01% Tween-20 (Pierce Chemical)), 50 pM control oligonucleotide B2 (Affymetrix, Santa Clara, CA), 0.1 mg/ml Herring Sperm DNA (Promega, Madison, WI Corporation), 0.5 mg/ml acetylated BSA (Invitrogen, Carlsbad, CA) and 1 X Eukaryotic Hybridization Controls (bioB, bioC, bioD, and cre at 1.5, 5, 25 and 100 pM respectively) (Affymetrix, Santa Clara, CA) for 16 hours at 45°C on a rotisserie at 60 rpm. Prior to application to the GeneChip®, samples were heated (95°C for 5 minutes), incubated (45°C for five minutes), and spun (14,000-x g for five minutes). Following hybridization, the labeled samples were removed from the GeneChip®, stored in the appropriate vial at –20C, and filled with non-stringent buffer A containing 6X SSPE (Ambion, Austin, TX), 0.01% Tween 20. All GeneChips® were post processed using the automated Affymetrix GeneChip® Fluidics Station 450. The post processing protocol of the Rat 2.0 Genome GeneChip® included: one wash (10 cycles with 2 mixes/cycle) of non-stringent buffer A at 25°C; one wash of 4 cycles comprised of 15 mixes/cycle with stringent buffer B (100mM MES, 0.1 M NaCl, 0.01% Tween 20) at 50C; staining of the probe array for 10 minutes at 25C in Streptavidin-Phycoerythrin (SAPE) solution (1X MES stain buffer (100 mM MES, 1 M NaCl, 0.05% Tween 20), 2 mg/ml acetylated BSA, 10 µg/ml SAPE (Molecular Probes); post staining consisting of 10 wash cycles of 4 mixes/cycle with non-stringent buffer A at 25°C; a second staining of the probe array for 10 minutes in antibody solution (1X MES stain buffer, 2 mg/ml acetylated BSA, 0.1 mg/ml Normal Goat IgG (Sigma-Aldrich, St. Louis, MO), 3 µg/ml biotinylated antibody (Vector Laboratories); a third stain of the probe array for 10 minutes in SAPE solution at 25C; and a final wash- 15 cycles of 4 mixes/cycle with non-stringent buffer A at 30°C.
Sample_scan_protocol	To quantify flourescence from each feature on the GeneChip, all GeneChips were scanned at a wavelength of 570 nm, using the GC3000 G7 scanner. Flourescent signals corresponding to hybridization intensities were analyzed via Affymetrix GCOS software
Sample_data_processing	Quantification of GeneChips was performed by Affymetrix GeneChip Operating Software (AGCC version 2.0).
Sample_data_processing	The data were compiled with EzArray using Affymetrix default analysis settings and global scaling as normalization method. Data were analyzed using the two-sample Welch t-test to determine significance per gene. To minimize the false discovery rate, the Storey q-value method was performed and significance was based on q < .01. Any Q-value less than or equal to 0.01 per gene was considered significant. Paired t-tests were used to evaluate differences between intact and day 3, intact and day 7, or day 3 and day 7. A critical value of .05 was considered as the criterion to select a significant fold change in gene expression between days. Additionally, microarray data were log transformed [log (base 2)] and the log ratio between the intact ligament with day 3 or day 7 was calculated and expressed as fold change (e.g. (mean day 3-mean intact control)= day 3 difference; fold change=2^(day 3 difference). A gene was considered up or down regulated if the day 3 or day 7 samples were at least 2 fold different than the intact controls.Data are presented as fold change of the means of day 3 and day 7 from the intact ligament values (fold change ± S.D.). Microarray data were functionally grouped using the Database for Annotation, Visualization, and Integrated Discovery (D.A.V.I.D.; http://david.abcc.ncifcrf.gov), integrating functional genomic annotations with intuitive graphical summaries. Using the Affymetrix probes as identifiers, the samples were subjected to functional annotation clustering based on function and gene ontology (GO). The categories of interest were further based on the enrichment score (ES) of the gene group. The ES, determined by the minus log transformation of the geometric mean p-values from all enriched annotation terms, ranks the biological significance of a gene cluster based on the scores of all the annotation terms.
Sample_platform_id	GPL1355
Sample_contact_name	Ray,,Vanderby
Sample_contact_laboratory	WIMR Room 5068
Sample_contact_department	Orthopedics and Rehabilitation
Sample_contact_institute	University of Wisconsin
Sample_contact_address	1111 Highland Ave
Sample_contact_city	Madison
Sample_contact_state	WI
Sample_contact_zip/postal_code	53705
Sample_contact_country	USA
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1154nnn/GSM1154497/suppl/GSM1154497_d3_7_9_left_RG230_2.0_110707.CEL.gz
Sample_series_id	GSE47676
Sample_data_row_count	31099

GSM1154498

GPL1355

MCL-Injured-Day 7-Rep 1

MCL-injured_collected at day 7 post-injury

strain: Wistar gender: male weight: 275-299g tissue: medial collateral ligament (MCL) sample group: day 7 post-injury

SAMPLE 4

Sample_geo_accession	GSM1154498
Sample_status	Public on Jun 06 2013
Sample_submission_date	Jun 05 2013
Sample_last_update_date	Jun 06 2013
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Rattus norvegicus
Sample_taxid_ch1	10116
Sample_treatment_protocol_ch1	Animals were subjected to bilateral MCL transections. At day 3 (n=9) or 7 post-injury (n=9), tissue was collected and used for microarray. Another 9 rats were used for intact MCL collection which served as the intact control.
Sample_growth_protocol_ch1	Ligaments were snap frozen in liquid nitrogen immediately upon removal from the body. Tissue was stored at -70C until use
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was isolated from the MCLs combining the TRIzol (Invitrogen, Carlsbad, CA) method with column fractionalization steps of the RNeasy Total RNA kit (Qiagen, Valencia, CA). Tissue was homogenized using a PowerGen 500 homogenizer (Fisher Scientific, Pittsburgh, PA). Subsequent to RNA isolation, yield and purity of RNA was quantified by nanodrop spectrophotometric measurement at 260 nm absorbance (Nanodrop Technologies, Wilmington, DE). Total RNA was converted to double-stranded cDNA using the oligo dT primer containing the T7 RNA polymerase promoter (5’ – GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-T24-3’).
Sample_label_ch1	Biotin
Sample_label_protocol_ch1	Second strand synthesis was performed by incubation with 1x second strand buffer, 200 μM dNTPs, 10 U DNA Ligase (Invitrogen, Carlsbad, CA), 40 U DNA Polymerase I (Invitrogen, Carlsbad, CA), and 2 U RNaseH (Invitrogen, Carlsbad, CA) for two hours at 16°C followed by a five minute incubation with 10 U T4 Polymerase (Invitrogen, Carlsbad, CA). Double stranded cDNA was purified via GeneChip Sample Cleanup Module kit (Affymetrix, Santa Clara, CA Inc.). The biotin-labeled in vitro transcription reaction was performed using the cDNA template and the Affymetrix 3’-Amplification IVT Labeling kit. Briefly, double stranded cDNA was incubated with 1x IVT Labeling buffer, 1x Labeling NTP mix, and 1x T7 Labeling Enzyme mix for 16 hours (37°C) to yield cRNA. The cRNA was purified using the GeneChip Sample Cleanup Module. cRNA yield was determined by absorbance of 260 nM. The cRNA was fragmented to a 0.5 µg/µl final concentration in 1x fragmentation buffer (40 mM Tris-acetate, pH 8.1, 100 mM potassium acetate, and 30 mM magnesium acetate) for 32 minutes at 94°C. The sizeof the cRNA before (0.1 kb and longer) and after (35-200 base fragments) was checked by agarose gel electrophoresis.
Sample_hyb_protocol	GeneChips® (Affymetrix, Santa Clara, CA) were hybridized with biotin labeled cRNA in 300 µl, in 1 X Hybridization buffer (100 mM MES, 1M NaCl (Ambion, Austin, TX), 20 mM EDTA (Ambion, Austin, TX), and 0.01% Tween-20 (Pierce Chemical)), 50 pM control oligonucleotide B2 (Affymetrix, Santa Clara, CA), 0.1 mg/ml Herring Sperm DNA (Promega, Madison, WI Corporation), 0.5 mg/ml acetylated BSA (Invitrogen, Carlsbad, CA) and 1 X Eukaryotic Hybridization Controls (bioB, bioC, bioD, and cre at 1.5, 5, 25 and 100 pM respectively) (Affymetrix, Santa Clara, CA) for 16 hours at 45°C on a rotisserie at 60 rpm. Prior to application to the GeneChip®, samples were heated (95°C for 5 minutes), incubated (45°C for five minutes), and spun (14,000-x g for five minutes). Following hybridization, the labeled samples were removed from the GeneChip®, stored in the appropriate vial at –20C, and filled with non-stringent buffer A containing 6X SSPE (Ambion, Austin, TX), 0.01% Tween 20. All GeneChips® were post processed using the automated Affymetrix GeneChip® Fluidics Station 450. The post processing protocol of the Rat 2.0 Genome GeneChip® included: one wash (10 cycles with 2 mixes/cycle) of non-stringent buffer A at 25°C; one wash of 4 cycles comprised of 15 mixes/cycle with stringent buffer B (100mM MES, 0.1 M NaCl, 0.01% Tween 20) at 50C; staining of the probe array for 10 minutes at 25C in Streptavidin-Phycoerythrin (SAPE) solution (1X MES stain buffer (100 mM MES, 1 M NaCl, 0.05% Tween 20), 2 mg/ml acetylated BSA, 10 µg/ml SAPE (Molecular Probes); post staining consisting of 10 wash cycles of 4 mixes/cycle with non-stringent buffer A at 25°C; a second staining of the probe array for 10 minutes in antibody solution (1X MES stain buffer, 2 mg/ml acetylated BSA, 0.1 mg/ml Normal Goat IgG (Sigma-Aldrich, St. Louis, MO), 3 µg/ml biotinylated antibody (Vector Laboratories); a third stain of the probe array for 10 minutes in SAPE solution at 25C; and a final wash- 15 cycles of 4 mixes/cycle with non-stringent buffer A at 30°C.
Sample_scan_protocol	To quantify flourescence from each feature on the GeneChip, all GeneChips were scanned at a wavelength of 570 nm, using the GC3000 G7 scanner. Flourescent signals corresponding to hybridization intensities were analyzed via Affymetrix GCOS software
Sample_data_processing	Quantification of GeneChips was performed by Affymetrix GeneChip Operating Software (AGCC version 2.0).
Sample_data_processing	The data were compiled with EzArray using Affymetrix default analysis settings and global scaling as normalization method. Data were analyzed using the two-sample Welch t-test to determine significance per gene. To minimize the false discovery rate, the Storey q-value method was performed and significance was based on q < .01. Any Q-value less than or equal to 0.01 per gene was considered significant. Paired t-tests were used to evaluate differences between intact and day 3, intact and day 7, or day 3 and day 7. A critical value of .05 was considered as the criterion to select a significant fold change in gene expression between days. Additionally, microarray data were log transformed [log (base 2)] and the log ratio between the intact ligament with day 3 or day 7 was calculated and expressed as fold change (e.g. (mean day 3-mean intact control)= day 3 difference; fold change=2^(day 3 difference). A gene was considered up or down regulated if the day 3 or day 7 samples were at least 2 fold different than the intact controls.Data are presented as fold change of the means of day 3 and day 7 from the intact ligament values (fold change ± S.D.). Microarray data were functionally grouped using the Database for Annotation, Visualization, and Integrated Discovery (D.A.V.I.D.; http://david.abcc.ncifcrf.gov), integrating functional genomic annotations with intuitive graphical summaries. Using the Affymetrix probes as identifiers, the samples were subjected to functional annotation clustering based on function and gene ontology (GO). The categories of interest were further based on the enrichment score (ES) of the gene group. The ES, determined by the minus log transformation of the geometric mean p-values from all enriched annotation terms, ranks the biological significance of a gene cluster based on the scores of all the annotation terms.
Sample_platform_id	GPL1355
Sample_contact_name	Ray,,Vanderby
Sample_contact_laboratory	WIMR Room 5068
Sample_contact_department	Orthopedics and Rehabilitation
Sample_contact_institute	University of Wisconsin
Sample_contact_address	1111 Highland Ave
Sample_contact_city	Madison
Sample_contact_state	WI
Sample_contact_zip/postal_code	53705
Sample_contact_country	USA
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1154nnn/GSM1154498/suppl/GSM1154498_d7_1_3_RG230_2.0_110707.CEL.gz
Sample_series_id	GSE47676
Sample_data_row_count	31099

GSM1154499

GPL1355

MCL-Injured-Day 7-Rep 2

MCL-injured_collected at day 7 post-injury

strain: Wistar gender: male weight: 275-299g tissue: medial collateral ligament (MCL) sample group: day 7 post-injury

SAMPLE 5

Sample_geo_accession	GSM1154499
Sample_status	Public on Jun 06 2013
Sample_submission_date	Jun 05 2013
Sample_last_update_date	Jun 06 2013
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Rattus norvegicus
Sample_taxid_ch1	10116
Sample_treatment_protocol_ch1	Animals were subjected to bilateral MCL transections. At day 3 (n=9) or 7 post-injury (n=9), tissue was collected and used for microarray. Another 9 rats were used for intact MCL collection which served as the intact control.
Sample_growth_protocol_ch1	Ligaments were snap frozen in liquid nitrogen immediately upon removal from the body. Tissue was stored at -70C until use
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was isolated from the MCLs combining the TRIzol (Invitrogen, Carlsbad, CA) method with column fractionalization steps of the RNeasy Total RNA kit (Qiagen, Valencia, CA). Tissue was homogenized using a PowerGen 500 homogenizer (Fisher Scientific, Pittsburgh, PA). Subsequent to RNA isolation, yield and purity of RNA was quantified by nanodrop spectrophotometric measurement at 260 nm absorbance (Nanodrop Technologies, Wilmington, DE). Total RNA was converted to double-stranded cDNA using the oligo dT primer containing the T7 RNA polymerase promoter (5’ – GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-T24-3’).
Sample_label_ch1	Biotin
Sample_label_protocol_ch1	Second strand synthesis was performed by incubation with 1x second strand buffer, 200 μM dNTPs, 10 U DNA Ligase (Invitrogen, Carlsbad, CA), 40 U DNA Polymerase I (Invitrogen, Carlsbad, CA), and 2 U RNaseH (Invitrogen, Carlsbad, CA) for two hours at 16°C followed by a five minute incubation with 10 U T4 Polymerase (Invitrogen, Carlsbad, CA). Double stranded cDNA was purified via GeneChip Sample Cleanup Module kit (Affymetrix, Santa Clara, CA Inc.). The biotin-labeled in vitro transcription reaction was performed using the cDNA template and the Affymetrix 3’-Amplification IVT Labeling kit. Briefly, double stranded cDNA was incubated with 1x IVT Labeling buffer, 1x Labeling NTP mix, and 1x T7 Labeling Enzyme mix for 16 hours (37°C) to yield cRNA. The cRNA was purified using the GeneChip Sample Cleanup Module. cRNA yield was determined by absorbance of 260 nM. The cRNA was fragmented to a 0.5 µg/µl final concentration in 1x fragmentation buffer (40 mM Tris-acetate, pH 8.1, 100 mM potassium acetate, and 30 mM magnesium acetate) for 32 minutes at 94°C. The sizeof the cRNA before (0.1 kb and longer) and after (35-200 base fragments) was checked by agarose gel electrophoresis.
Sample_hyb_protocol	GeneChips® (Affymetrix, Santa Clara, CA) were hybridized with biotin labeled cRNA in 300 µl, in 1 X Hybridization buffer (100 mM MES, 1M NaCl (Ambion, Austin, TX), 20 mM EDTA (Ambion, Austin, TX), and 0.01% Tween-20 (Pierce Chemical)), 50 pM control oligonucleotide B2 (Affymetrix, Santa Clara, CA), 0.1 mg/ml Herring Sperm DNA (Promega, Madison, WI Corporation), 0.5 mg/ml acetylated BSA (Invitrogen, Carlsbad, CA) and 1 X Eukaryotic Hybridization Controls (bioB, bioC, bioD, and cre at 1.5, 5, 25 and 100 pM respectively) (Affymetrix, Santa Clara, CA) for 16 hours at 45°C on a rotisserie at 60 rpm. Prior to application to the GeneChip®, samples were heated (95°C for 5 minutes), incubated (45°C for five minutes), and spun (14,000-x g for five minutes). Following hybridization, the labeled samples were removed from the GeneChip®, stored in the appropriate vial at –20C, and filled with non-stringent buffer A containing 6X SSPE (Ambion, Austin, TX), 0.01% Tween 20. All GeneChips® were post processed using the automated Affymetrix GeneChip® Fluidics Station 450. The post processing protocol of the Rat 2.0 Genome GeneChip® included: one wash (10 cycles with 2 mixes/cycle) of non-stringent buffer A at 25°C; one wash of 4 cycles comprised of 15 mixes/cycle with stringent buffer B (100mM MES, 0.1 M NaCl, 0.01% Tween 20) at 50C; staining of the probe array for 10 minutes at 25C in Streptavidin-Phycoerythrin (SAPE) solution (1X MES stain buffer (100 mM MES, 1 M NaCl, 0.05% Tween 20), 2 mg/ml acetylated BSA, 10 µg/ml SAPE (Molecular Probes); post staining consisting of 10 wash cycles of 4 mixes/cycle with non-stringent buffer A at 25°C; a second staining of the probe array for 10 minutes in antibody solution (1X MES stain buffer, 2 mg/ml acetylated BSA, 0.1 mg/ml Normal Goat IgG (Sigma-Aldrich, St. Louis, MO), 3 µg/ml biotinylated antibody (Vector Laboratories); a third stain of the probe array for 10 minutes in SAPE solution at 25C; and a final wash- 15 cycles of 4 mixes/cycle with non-stringent buffer A at 30°C.
Sample_scan_protocol	To quantify flourescence from each feature on the GeneChip, all GeneChips were scanned at a wavelength of 570 nm, using the GC3000 G7 scanner. Flourescent signals corresponding to hybridization intensities were analyzed via Affymetrix GCOS software
Sample_data_processing	Quantification of GeneChips was performed by Affymetrix GeneChip Operating Software (AGCC version 2.0).
Sample_data_processing	The data were compiled with EzArray using Affymetrix default analysis settings and global scaling as normalization method. Data were analyzed using the two-sample Welch t-test to determine significance per gene. To minimize the false discovery rate, the Storey q-value method was performed and significance was based on q < .01. Any Q-value less than or equal to 0.01 per gene was considered significant. Paired t-tests were used to evaluate differences between intact and day 3, intact and day 7, or day 3 and day 7. A critical value of .05 was considered as the criterion to select a significant fold change in gene expression between days. Additionally, microarray data were log transformed [log (base 2)] and the log ratio between the intact ligament with day 3 or day 7 was calculated and expressed as fold change (e.g. (mean day 3-mean intact control)= day 3 difference; fold change=2^(day 3 difference). A gene was considered up or down regulated if the day 3 or day 7 samples were at least 2 fold different than the intact controls.Data are presented as fold change of the means of day 3 and day 7 from the intact ligament values (fold change ± S.D.). Microarray data were functionally grouped using the Database for Annotation, Visualization, and Integrated Discovery (D.A.V.I.D.; http://david.abcc.ncifcrf.gov), integrating functional genomic annotations with intuitive graphical summaries. Using the Affymetrix probes as identifiers, the samples were subjected to functional annotation clustering based on function and gene ontology (GO). The categories of interest were further based on the enrichment score (ES) of the gene group. The ES, determined by the minus log transformation of the geometric mean p-values from all enriched annotation terms, ranks the biological significance of a gene cluster based on the scores of all the annotation terms.
Sample_platform_id	GPL1355
Sample_contact_name	Ray,,Vanderby
Sample_contact_laboratory	WIMR Room 5068
Sample_contact_department	Orthopedics and Rehabilitation
Sample_contact_institute	University of Wisconsin
Sample_contact_address	1111 Highland Ave
Sample_contact_city	Madison
Sample_contact_state	WI
Sample_contact_zip/postal_code	53705
Sample_contact_country	USA
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1154nnn/GSM1154499/suppl/GSM1154499_d7_4_6_RG230_2.0_110707.CEL.gz
Sample_series_id	GSE47676
Sample_data_row_count	31099

GSM1154500

GPL1355

MCL-Injured-Day 7-Rep 3

MCL-injured_collected at day 7 post-injury

strain: Wistar gender: male weight: 275-299g tissue: medial collateral ligament (MCL) sample group: day 7 post-injury

SAMPLE 6

Sample_geo_accession	GSM1154500
Sample_status	Public on Jun 06 2013
Sample_submission_date	Jun 05 2013
Sample_last_update_date	Jun 06 2013
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Rattus norvegicus
Sample_taxid_ch1	10116
Sample_treatment_protocol_ch1	Animals were subjected to bilateral MCL transections. At day 3 (n=9) or 7 post-injury (n=9), tissue was collected and used for microarray. Another 9 rats were used for intact MCL collection which served as the intact control.
Sample_growth_protocol_ch1	Ligaments were snap frozen in liquid nitrogen immediately upon removal from the body. Tissue was stored at -70C until use
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was isolated from the MCLs combining the TRIzol (Invitrogen, Carlsbad, CA) method with column fractionalization steps of the RNeasy Total RNA kit (Qiagen, Valencia, CA). Tissue was homogenized using a PowerGen 500 homogenizer (Fisher Scientific, Pittsburgh, PA). Subsequent to RNA isolation, yield and purity of RNA was quantified by nanodrop spectrophotometric measurement at 260 nm absorbance (Nanodrop Technologies, Wilmington, DE). Total RNA was converted to double-stranded cDNA using the oligo dT primer containing the T7 RNA polymerase promoter (5’ – GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-T24-3’).
Sample_label_ch1	Biotin
Sample_label_protocol_ch1	Second strand synthesis was performed by incubation with 1x second strand buffer, 200 μM dNTPs, 10 U DNA Ligase (Invitrogen, Carlsbad, CA), 40 U DNA Polymerase I (Invitrogen, Carlsbad, CA), and 2 U RNaseH (Invitrogen, Carlsbad, CA) for two hours at 16°C followed by a five minute incubation with 10 U T4 Polymerase (Invitrogen, Carlsbad, CA). Double stranded cDNA was purified via GeneChip Sample Cleanup Module kit (Affymetrix, Santa Clara, CA Inc.). The biotin-labeled in vitro transcription reaction was performed using the cDNA template and the Affymetrix 3’-Amplification IVT Labeling kit. Briefly, double stranded cDNA was incubated with 1x IVT Labeling buffer, 1x Labeling NTP mix, and 1x T7 Labeling Enzyme mix for 16 hours (37°C) to yield cRNA. The cRNA was purified using the GeneChip Sample Cleanup Module. cRNA yield was determined by absorbance of 260 nM. The cRNA was fragmented to a 0.5 µg/µl final concentration in 1x fragmentation buffer (40 mM Tris-acetate, pH 8.1, 100 mM potassium acetate, and 30 mM magnesium acetate) for 32 minutes at 94°C. The sizeof the cRNA before (0.1 kb and longer) and after (35-200 base fragments) was checked by agarose gel electrophoresis.
Sample_hyb_protocol	GeneChips® (Affymetrix, Santa Clara, CA) were hybridized with biotin labeled cRNA in 300 µl, in 1 X Hybridization buffer (100 mM MES, 1M NaCl (Ambion, Austin, TX), 20 mM EDTA (Ambion, Austin, TX), and 0.01% Tween-20 (Pierce Chemical)), 50 pM control oligonucleotide B2 (Affymetrix, Santa Clara, CA), 0.1 mg/ml Herring Sperm DNA (Promega, Madison, WI Corporation), 0.5 mg/ml acetylated BSA (Invitrogen, Carlsbad, CA) and 1 X Eukaryotic Hybridization Controls (bioB, bioC, bioD, and cre at 1.5, 5, 25 and 100 pM respectively) (Affymetrix, Santa Clara, CA) for 16 hours at 45°C on a rotisserie at 60 rpm. Prior to application to the GeneChip®, samples were heated (95°C for 5 minutes), incubated (45°C for five minutes), and spun (14,000-x g for five minutes). Following hybridization, the labeled samples were removed from the GeneChip®, stored in the appropriate vial at –20C, and filled with non-stringent buffer A containing 6X SSPE (Ambion, Austin, TX), 0.01% Tween 20. All GeneChips® were post processed using the automated Affymetrix GeneChip® Fluidics Station 450. The post processing protocol of the Rat 2.0 Genome GeneChip® included: one wash (10 cycles with 2 mixes/cycle) of non-stringent buffer A at 25°C; one wash of 4 cycles comprised of 15 mixes/cycle with stringent buffer B (100mM MES, 0.1 M NaCl, 0.01% Tween 20) at 50C; staining of the probe array for 10 minutes at 25C in Streptavidin-Phycoerythrin (SAPE) solution (1X MES stain buffer (100 mM MES, 1 M NaCl, 0.05% Tween 20), 2 mg/ml acetylated BSA, 10 µg/ml SAPE (Molecular Probes); post staining consisting of 10 wash cycles of 4 mixes/cycle with non-stringent buffer A at 25°C; a second staining of the probe array for 10 minutes in antibody solution (1X MES stain buffer, 2 mg/ml acetylated BSA, 0.1 mg/ml Normal Goat IgG (Sigma-Aldrich, St. Louis, MO), 3 µg/ml biotinylated antibody (Vector Laboratories); a third stain of the probe array for 10 minutes in SAPE solution at 25C; and a final wash- 15 cycles of 4 mixes/cycle with non-stringent buffer A at 30°C.
Sample_scan_protocol	To quantify flourescence from each feature on the GeneChip, all GeneChips were scanned at a wavelength of 570 nm, using the GC3000 G7 scanner. Flourescent signals corresponding to hybridization intensities were analyzed via Affymetrix GCOS software
Sample_data_processing	Quantification of GeneChips was performed by Affymetrix GeneChip Operating Software (AGCC version 2.0).
Sample_data_processing	The data were compiled with EzArray using Affymetrix default analysis settings and global scaling as normalization method. Data were analyzed using the two-sample Welch t-test to determine significance per gene. To minimize the false discovery rate, the Storey q-value method was performed and significance was based on q < .01. Any Q-value less than or equal to 0.01 per gene was considered significant. Paired t-tests were used to evaluate differences between intact and day 3, intact and day 7, or day 3 and day 7. A critical value of .05 was considered as the criterion to select a significant fold change in gene expression between days. Additionally, microarray data were log transformed [log (base 2)] and the log ratio between the intact ligament with day 3 or day 7 was calculated and expressed as fold change (e.g. (mean day 3-mean intact control)= day 3 difference; fold change=2^(day 3 difference). A gene was considered up or down regulated if the day 3 or day 7 samples were at least 2 fold different than the intact controls.Data are presented as fold change of the means of day 3 and day 7 from the intact ligament values (fold change ± S.D.). Microarray data were functionally grouped using the Database for Annotation, Visualization, and Integrated Discovery (D.A.V.I.D.; http://david.abcc.ncifcrf.gov), integrating functional genomic annotations with intuitive graphical summaries. Using the Affymetrix probes as identifiers, the samples were subjected to functional annotation clustering based on function and gene ontology (GO). The categories of interest were further based on the enrichment score (ES) of the gene group. The ES, determined by the minus log transformation of the geometric mean p-values from all enriched annotation terms, ranks the biological significance of a gene cluster based on the scores of all the annotation terms.
Sample_platform_id	GPL1355
Sample_contact_name	Ray,,Vanderby
Sample_contact_laboratory	WIMR Room 5068
Sample_contact_department	Orthopedics and Rehabilitation
Sample_contact_institute	University of Wisconsin
Sample_contact_address	1111 Highland Ave
Sample_contact_city	Madison
Sample_contact_state	WI
Sample_contact_zip/postal_code	53705
Sample_contact_country	USA
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1154nnn/GSM1154500/suppl/GSM1154500_d7_7_9_RG230_2.0_110707.CEL.gz
Sample_series_id	GSE47676
Sample_data_row_count	31099

GSM1154501

GPL1355

MCL-No Injury CX-Rep 1

MCL-no injury-intact control

strain: Wistar gender: male weight: 275-299g tissue: medial collateral ligament (MCL) sample group: no injury-intact control

SAMPLE 7

Sample_geo_accession	GSM1154501
Sample_status	Public on Jun 06 2013
Sample_submission_date	Jun 05 2013
Sample_last_update_date	Jun 06 2013
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Rattus norvegicus
Sample_taxid_ch1	10116
Sample_treatment_protocol_ch1	Animals were subjected to bilateral MCL transections. At day 3 (n=9) or 7 post-injury (n=9), tissue was collected and used for microarray. Another 9 rats were used for intact MCL collection which served as the intact control.
Sample_growth_protocol_ch1	Ligaments were snap frozen in liquid nitrogen immediately upon removal from the body. Tissue was stored at -70C until use
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was isolated from the MCLs combining the TRIzol (Invitrogen, Carlsbad, CA) method with column fractionalization steps of the RNeasy Total RNA kit (Qiagen, Valencia, CA). Tissue was homogenized using a PowerGen 500 homogenizer (Fisher Scientific, Pittsburgh, PA). Subsequent to RNA isolation, yield and purity of RNA was quantified by nanodrop spectrophotometric measurement at 260 nm absorbance (Nanodrop Technologies, Wilmington, DE). Total RNA was converted to double-stranded cDNA using the oligo dT primer containing the T7 RNA polymerase promoter (5’ – GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-T24-3’).
Sample_label_ch1	Biotin
Sample_label_protocol_ch1	Second strand synthesis was performed by incubation with 1x second strand buffer, 200 μM dNTPs, 10 U DNA Ligase (Invitrogen, Carlsbad, CA), 40 U DNA Polymerase I (Invitrogen, Carlsbad, CA), and 2 U RNaseH (Invitrogen, Carlsbad, CA) for two hours at 16°C followed by a five minute incubation with 10 U T4 Polymerase (Invitrogen, Carlsbad, CA). Double stranded cDNA was purified via GeneChip Sample Cleanup Module kit (Affymetrix, Santa Clara, CA Inc.). The biotin-labeled in vitro transcription reaction was performed using the cDNA template and the Affymetrix 3’-Amplification IVT Labeling kit. Briefly, double stranded cDNA was incubated with 1x IVT Labeling buffer, 1x Labeling NTP mix, and 1x T7 Labeling Enzyme mix for 16 hours (37°C) to yield cRNA. The cRNA was purified using the GeneChip Sample Cleanup Module. cRNA yield was determined by absorbance of 260 nM. The cRNA was fragmented to a 0.5 µg/µl final concentration in 1x fragmentation buffer (40 mM Tris-acetate, pH 8.1, 100 mM potassium acetate, and 30 mM magnesium acetate) for 32 minutes at 94°C. The sizeof the cRNA before (0.1 kb and longer) and after (35-200 base fragments) was checked by agarose gel electrophoresis.
Sample_hyb_protocol	GeneChips® (Affymetrix, Santa Clara, CA) were hybridized with biotin labeled cRNA in 300 µl, in 1 X Hybridization buffer (100 mM MES, 1M NaCl (Ambion, Austin, TX), 20 mM EDTA (Ambion, Austin, TX), and 0.01% Tween-20 (Pierce Chemical)), 50 pM control oligonucleotide B2 (Affymetrix, Santa Clara, CA), 0.1 mg/ml Herring Sperm DNA (Promega, Madison, WI Corporation), 0.5 mg/ml acetylated BSA (Invitrogen, Carlsbad, CA) and 1 X Eukaryotic Hybridization Controls (bioB, bioC, bioD, and cre at 1.5, 5, 25 and 100 pM respectively) (Affymetrix, Santa Clara, CA) for 16 hours at 45°C on a rotisserie at 60 rpm. Prior to application to the GeneChip®, samples were heated (95°C for 5 minutes), incubated (45°C for five minutes), and spun (14,000-x g for five minutes). Following hybridization, the labeled samples were removed from the GeneChip®, stored in the appropriate vial at –20C, and filled with non-stringent buffer A containing 6X SSPE (Ambion, Austin, TX), 0.01% Tween 20. All GeneChips® were post processed using the automated Affymetrix GeneChip® Fluidics Station 450. The post processing protocol of the Rat 2.0 Genome GeneChip® included: one wash (10 cycles with 2 mixes/cycle) of non-stringent buffer A at 25°C; one wash of 4 cycles comprised of 15 mixes/cycle with stringent buffer B (100mM MES, 0.1 M NaCl, 0.01% Tween 20) at 50C; staining of the probe array for 10 minutes at 25C in Streptavidin-Phycoerythrin (SAPE) solution (1X MES stain buffer (100 mM MES, 1 M NaCl, 0.05% Tween 20), 2 mg/ml acetylated BSA, 10 µg/ml SAPE (Molecular Probes); post staining consisting of 10 wash cycles of 4 mixes/cycle with non-stringent buffer A at 25°C; a second staining of the probe array for 10 minutes in antibody solution (1X MES stain buffer, 2 mg/ml acetylated BSA, 0.1 mg/ml Normal Goat IgG (Sigma-Aldrich, St. Louis, MO), 3 µg/ml biotinylated antibody (Vector Laboratories); a third stain of the probe array for 10 minutes in SAPE solution at 25C; and a final wash- 15 cycles of 4 mixes/cycle with non-stringent buffer A at 30°C.
Sample_scan_protocol	To quantify flourescence from each feature on the GeneChip, all GeneChips were scanned at a wavelength of 570 nm, using the GC3000 G7 scanner. Flourescent signals corresponding to hybridization intensities were analyzed via Affymetrix GCOS software
Sample_data_processing	Quantification of GeneChips was performed by Affymetrix GeneChip Operating Software (AGCC version 2.0).
Sample_data_processing	The data were compiled with EzArray using Affymetrix default analysis settings and global scaling as normalization method. Data were analyzed using the two-sample Welch t-test to determine significance per gene. To minimize the false discovery rate, the Storey q-value method was performed and significance was based on q < .01. Any Q-value less than or equal to 0.01 per gene was considered significant. Paired t-tests were used to evaluate differences between intact and day 3, intact and day 7, or day 3 and day 7. A critical value of .05 was considered as the criterion to select a significant fold change in gene expression between days. Additionally, microarray data were log transformed [log (base 2)] and the log ratio between the intact ligament with day 3 or day 7 was calculated and expressed as fold change (e.g. (mean day 3-mean intact control)= day 3 difference; fold change=2^(day 3 difference). A gene was considered up or down regulated if the day 3 or day 7 samples were at least 2 fold different than the intact controls.Data are presented as fold change of the means of day 3 and day 7 from the intact ligament values (fold change ± S.D.). Microarray data were functionally grouped using the Database for Annotation, Visualization, and Integrated Discovery (D.A.V.I.D.; http://david.abcc.ncifcrf.gov), integrating functional genomic annotations with intuitive graphical summaries. Using the Affymetrix probes as identifiers, the samples were subjected to functional annotation clustering based on function and gene ontology (GO). The categories of interest were further based on the enrichment score (ES) of the gene group. The ES, determined by the minus log transformation of the geometric mean p-values from all enriched annotation terms, ranks the biological significance of a gene cluster based on the scores of all the annotation terms.
Sample_platform_id	GPL1355
Sample_contact_name	Ray,,Vanderby
Sample_contact_laboratory	WIMR Room 5068
Sample_contact_department	Orthopedics and Rehabilitation
Sample_contact_institute	University of Wisconsin
Sample_contact_address	1111 Highland Ave
Sample_contact_city	Madison
Sample_contact_state	WI
Sample_contact_zip/postal_code	53705
Sample_contact_country	USA
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1154nnn/GSM1154501/suppl/GSM1154501_norm_1_3_left_RG230_2.0_110707.CEL.gz
Sample_series_id	GSE47676
Sample_data_row_count	31099

GSM1154502

GPL1355

MCL-No Injury CX-Rep 2

MCL-no injury-intact control

strain: Wistar gender: male weight: 275-299g tissue: medial collateral ligament (MCL) sample group: no injury-intact control

SAMPLE 8

Sample_geo_accession	GSM1154502
Sample_status	Public on Jun 06 2013
Sample_submission_date	Jun 05 2013
Sample_last_update_date	Jun 06 2013
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Rattus norvegicus
Sample_taxid_ch1	10116
Sample_treatment_protocol_ch1	Animals were subjected to bilateral MCL transections. At day 3 (n=9) or 7 post-injury (n=9), tissue was collected and used for microarray. Another 9 rats were used for intact MCL collection which served as the intact control.
Sample_growth_protocol_ch1	Ligaments were snap frozen in liquid nitrogen immediately upon removal from the body. Tissue was stored at -70C until use
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was isolated from the MCLs combining the TRIzol (Invitrogen, Carlsbad, CA) method with column fractionalization steps of the RNeasy Total RNA kit (Qiagen, Valencia, CA). Tissue was homogenized using a PowerGen 500 homogenizer (Fisher Scientific, Pittsburgh, PA). Subsequent to RNA isolation, yield and purity of RNA was quantified by nanodrop spectrophotometric measurement at 260 nm absorbance (Nanodrop Technologies, Wilmington, DE). Total RNA was converted to double-stranded cDNA using the oligo dT primer containing the T7 RNA polymerase promoter (5’ – GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-T24-3’).
Sample_label_ch1	Biotin
Sample_label_protocol_ch1	Second strand synthesis was performed by incubation with 1x second strand buffer, 200 μM dNTPs, 10 U DNA Ligase (Invitrogen, Carlsbad, CA), 40 U DNA Polymerase I (Invitrogen, Carlsbad, CA), and 2 U RNaseH (Invitrogen, Carlsbad, CA) for two hours at 16°C followed by a five minute incubation with 10 U T4 Polymerase (Invitrogen, Carlsbad, CA). Double stranded cDNA was purified via GeneChip Sample Cleanup Module kit (Affymetrix, Santa Clara, CA Inc.). The biotin-labeled in vitro transcription reaction was performed using the cDNA template and the Affymetrix 3’-Amplification IVT Labeling kit. Briefly, double stranded cDNA was incubated with 1x IVT Labeling buffer, 1x Labeling NTP mix, and 1x T7 Labeling Enzyme mix for 16 hours (37°C) to yield cRNA. The cRNA was purified using the GeneChip Sample Cleanup Module. cRNA yield was determined by absorbance of 260 nM. The cRNA was fragmented to a 0.5 µg/µl final concentration in 1x fragmentation buffer (40 mM Tris-acetate, pH 8.1, 100 mM potassium acetate, and 30 mM magnesium acetate) for 32 minutes at 94°C. The sizeof the cRNA before (0.1 kb and longer) and after (35-200 base fragments) was checked by agarose gel electrophoresis.
Sample_hyb_protocol	GeneChips® (Affymetrix, Santa Clara, CA) were hybridized with biotin labeled cRNA in 300 µl, in 1 X Hybridization buffer (100 mM MES, 1M NaCl (Ambion, Austin, TX), 20 mM EDTA (Ambion, Austin, TX), and 0.01% Tween-20 (Pierce Chemical)), 50 pM control oligonucleotide B2 (Affymetrix, Santa Clara, CA), 0.1 mg/ml Herring Sperm DNA (Promega, Madison, WI Corporation), 0.5 mg/ml acetylated BSA (Invitrogen, Carlsbad, CA) and 1 X Eukaryotic Hybridization Controls (bioB, bioC, bioD, and cre at 1.5, 5, 25 and 100 pM respectively) (Affymetrix, Santa Clara, CA) for 16 hours at 45°C on a rotisserie at 60 rpm. Prior to application to the GeneChip®, samples were heated (95°C for 5 minutes), incubated (45°C for five minutes), and spun (14,000-x g for five minutes). Following hybridization, the labeled samples were removed from the GeneChip®, stored in the appropriate vial at –20C, and filled with non-stringent buffer A containing 6X SSPE (Ambion, Austin, TX), 0.01% Tween 20. All GeneChips® were post processed using the automated Affymetrix GeneChip® Fluidics Station 450. The post processing protocol of the Rat 2.0 Genome GeneChip® included: one wash (10 cycles with 2 mixes/cycle) of non-stringent buffer A at 25°C; one wash of 4 cycles comprised of 15 mixes/cycle with stringent buffer B (100mM MES, 0.1 M NaCl, 0.01% Tween 20) at 50C; staining of the probe array for 10 minutes at 25C in Streptavidin-Phycoerythrin (SAPE) solution (1X MES stain buffer (100 mM MES, 1 M NaCl, 0.05% Tween 20), 2 mg/ml acetylated BSA, 10 µg/ml SAPE (Molecular Probes); post staining consisting of 10 wash cycles of 4 mixes/cycle with non-stringent buffer A at 25°C; a second staining of the probe array for 10 minutes in antibody solution (1X MES stain buffer, 2 mg/ml acetylated BSA, 0.1 mg/ml Normal Goat IgG (Sigma-Aldrich, St. Louis, MO), 3 µg/ml biotinylated antibody (Vector Laboratories); a third stain of the probe array for 10 minutes in SAPE solution at 25C; and a final wash- 15 cycles of 4 mixes/cycle with non-stringent buffer A at 30°C.
Sample_scan_protocol	To quantify flourescence from each feature on the GeneChip, all GeneChips were scanned at a wavelength of 570 nm, using the GC3000 G7 scanner. Flourescent signals corresponding to hybridization intensities were analyzed via Affymetrix GCOS software
Sample_data_processing	Quantification of GeneChips was performed by Affymetrix GeneChip Operating Software (AGCC version 2.0).
Sample_data_processing	The data were compiled with EzArray using Affymetrix default analysis settings and global scaling as normalization method. Data were analyzed using the two-sample Welch t-test to determine significance per gene. To minimize the false discovery rate, the Storey q-value method was performed and significance was based on q < .01. Any Q-value less than or equal to 0.01 per gene was considered significant. Paired t-tests were used to evaluate differences between intact and day 3, intact and day 7, or day 3 and day 7. A critical value of .05 was considered as the criterion to select a significant fold change in gene expression between days. Additionally, microarray data were log transformed [log (base 2)] and the log ratio between the intact ligament with day 3 or day 7 was calculated and expressed as fold change (e.g. (mean day 3-mean intact control)= day 3 difference; fold change=2^(day 3 difference). A gene was considered up or down regulated if the day 3 or day 7 samples were at least 2 fold different than the intact controls.Data are presented as fold change of the means of day 3 and day 7 from the intact ligament values (fold change ± S.D.). Microarray data were functionally grouped using the Database for Annotation, Visualization, and Integrated Discovery (D.A.V.I.D.; http://david.abcc.ncifcrf.gov), integrating functional genomic annotations with intuitive graphical summaries. Using the Affymetrix probes as identifiers, the samples were subjected to functional annotation clustering based on function and gene ontology (GO). The categories of interest were further based on the enrichment score (ES) of the gene group. The ES, determined by the minus log transformation of the geometric mean p-values from all enriched annotation terms, ranks the biological significance of a gene cluster based on the scores of all the annotation terms.
Sample_platform_id	GPL1355
Sample_contact_name	Ray,,Vanderby
Sample_contact_laboratory	WIMR Room 5068
Sample_contact_department	Orthopedics and Rehabilitation
Sample_contact_institute	University of Wisconsin
Sample_contact_address	1111 Highland Ave
Sample_contact_city	Madison
Sample_contact_state	WI
Sample_contact_zip/postal_code	53705
Sample_contact_country	USA
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1154nnn/GSM1154502/suppl/GSM1154502_norm_4_6_left_RG230_2.0_110707.CEL.gz
Sample_series_id	GSE47676
Sample_data_row_count	31099

GSM1154503

GPL1355

MCL-No Injury CX-Rep 3

MCL-no injury-intact control

strain: Wistar gender: male weight: 275-299g tissue: medial collateral ligament (MCL) sample group: no injury-intact control

SAMPLE 9

Sample_geo_accession	GSM1154503
Sample_status	Public on Jun 06 2013
Sample_submission_date	Jun 05 2013
Sample_last_update_date	Jun 06 2013
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Rattus norvegicus
Sample_taxid_ch1	10116
Sample_treatment_protocol_ch1	Animals were subjected to bilateral MCL transections. At day 3 (n=9) or 7 post-injury (n=9), tissue was collected and used for microarray. Another 9 rats were used for intact MCL collection which served as the intact control.
Sample_growth_protocol_ch1	Ligaments were snap frozen in liquid nitrogen immediately upon removal from the body. Tissue was stored at -70C until use
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was isolated from the MCLs combining the TRIzol (Invitrogen, Carlsbad, CA) method with column fractionalization steps of the RNeasy Total RNA kit (Qiagen, Valencia, CA). Tissue was homogenized using a PowerGen 500 homogenizer (Fisher Scientific, Pittsburgh, PA). Subsequent to RNA isolation, yield and purity of RNA was quantified by nanodrop spectrophotometric measurement at 260 nm absorbance (Nanodrop Technologies, Wilmington, DE). Total RNA was converted to double-stranded cDNA using the oligo dT primer containing the T7 RNA polymerase promoter (5’ – GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-T24-3’).
Sample_label_ch1	Biotin
Sample_label_protocol_ch1	Second strand synthesis was performed by incubation with 1x second strand buffer, 200 μM dNTPs, 10 U DNA Ligase (Invitrogen, Carlsbad, CA), 40 U DNA Polymerase I (Invitrogen, Carlsbad, CA), and 2 U RNaseH (Invitrogen, Carlsbad, CA) for two hours at 16°C followed by a five minute incubation with 10 U T4 Polymerase (Invitrogen, Carlsbad, CA). Double stranded cDNA was purified via GeneChip Sample Cleanup Module kit (Affymetrix, Santa Clara, CA Inc.). The biotin-labeled in vitro transcription reaction was performed using the cDNA template and the Affymetrix 3’-Amplification IVT Labeling kit. Briefly, double stranded cDNA was incubated with 1x IVT Labeling buffer, 1x Labeling NTP mix, and 1x T7 Labeling Enzyme mix for 16 hours (37°C) to yield cRNA. The cRNA was purified using the GeneChip Sample Cleanup Module. cRNA yield was determined by absorbance of 260 nM. The cRNA was fragmented to a 0.5 µg/µl final concentration in 1x fragmentation buffer (40 mM Tris-acetate, pH 8.1, 100 mM potassium acetate, and 30 mM magnesium acetate) for 32 minutes at 94°C. The sizeof the cRNA before (0.1 kb and longer) and after (35-200 base fragments) was checked by agarose gel electrophoresis.
Sample_hyb_protocol	GeneChips® (Affymetrix, Santa Clara, CA) were hybridized with biotin labeled cRNA in 300 µl, in 1 X Hybridization buffer (100 mM MES, 1M NaCl (Ambion, Austin, TX), 20 mM EDTA (Ambion, Austin, TX), and 0.01% Tween-20 (Pierce Chemical)), 50 pM control oligonucleotide B2 (Affymetrix, Santa Clara, CA), 0.1 mg/ml Herring Sperm DNA (Promega, Madison, WI Corporation), 0.5 mg/ml acetylated BSA (Invitrogen, Carlsbad, CA) and 1 X Eukaryotic Hybridization Controls (bioB, bioC, bioD, and cre at 1.5, 5, 25 and 100 pM respectively) (Affymetrix, Santa Clara, CA) for 16 hours at 45°C on a rotisserie at 60 rpm. Prior to application to the GeneChip®, samples were heated (95°C for 5 minutes), incubated (45°C for five minutes), and spun (14,000-x g for five minutes). Following hybridization, the labeled samples were removed from the GeneChip®, stored in the appropriate vial at –20C, and filled with non-stringent buffer A containing 6X SSPE (Ambion, Austin, TX), 0.01% Tween 20. All GeneChips® were post processed using the automated Affymetrix GeneChip® Fluidics Station 450. The post processing protocol of the Rat 2.0 Genome GeneChip® included: one wash (10 cycles with 2 mixes/cycle) of non-stringent buffer A at 25°C; one wash of 4 cycles comprised of 15 mixes/cycle with stringent buffer B (100mM MES, 0.1 M NaCl, 0.01% Tween 20) at 50C; staining of the probe array for 10 minutes at 25C in Streptavidin-Phycoerythrin (SAPE) solution (1X MES stain buffer (100 mM MES, 1 M NaCl, 0.05% Tween 20), 2 mg/ml acetylated BSA, 10 µg/ml SAPE (Molecular Probes); post staining consisting of 10 wash cycles of 4 mixes/cycle with non-stringent buffer A at 25°C; a second staining of the probe array for 10 minutes in antibody solution (1X MES stain buffer, 2 mg/ml acetylated BSA, 0.1 mg/ml Normal Goat IgG (Sigma-Aldrich, St. Louis, MO), 3 µg/ml biotinylated antibody (Vector Laboratories); a third stain of the probe array for 10 minutes in SAPE solution at 25C; and a final wash- 15 cycles of 4 mixes/cycle with non-stringent buffer A at 30°C.
Sample_scan_protocol	To quantify flourescence from each feature on the GeneChip, all GeneChips were scanned at a wavelength of 570 nm, using the GC3000 G7 scanner. Flourescent signals corresponding to hybridization intensities were analyzed via Affymetrix GCOS software
Sample_data_processing	Quantification of GeneChips was performed by Affymetrix GeneChip Operating Software (AGCC version 2.0).
Sample_data_processing	The data were compiled with EzArray using Affymetrix default analysis settings and global scaling as normalization method. Data were analyzed using the two-sample Welch t-test to determine significance per gene. To minimize the false discovery rate, the Storey q-value method was performed and significance was based on q < .01. Any Q-value less than or equal to 0.01 per gene was considered significant. Paired t-tests were used to evaluate differences between intact and day 3, intact and day 7, or day 3 and day 7. A critical value of .05 was considered as the criterion to select a significant fold change in gene expression between days. Additionally, microarray data were log transformed [log (base 2)] and the log ratio between the intact ligament with day 3 or day 7 was calculated and expressed as fold change (e.g. (mean day 3-mean intact control)= day 3 difference; fold change=2^(day 3 difference). A gene was considered up or down regulated if the day 3 or day 7 samples were at least 2 fold different than the intact controls.Data are presented as fold change of the means of day 3 and day 7 from the intact ligament values (fold change ± S.D.). Microarray data were functionally grouped using the Database for Annotation, Visualization, and Integrated Discovery (D.A.V.I.D.; http://david.abcc.ncifcrf.gov), integrating functional genomic annotations with intuitive graphical summaries. Using the Affymetrix probes as identifiers, the samples were subjected to functional annotation clustering based on function and gene ontology (GO). The categories of interest were further based on the enrichment score (ES) of the gene group. The ES, determined by the minus log transformation of the geometric mean p-values from all enriched annotation terms, ranks the biological significance of a gene cluster based on the scores of all the annotation terms.
Sample_platform_id	GPL1355
Sample_contact_name	Ray,,Vanderby
Sample_contact_laboratory	WIMR Room 5068
Sample_contact_department	Orthopedics and Rehabilitation
Sample_contact_institute	University of Wisconsin
Sample_contact_address	1111 Highland Ave
Sample_contact_city	Madison
Sample_contact_state	WI
Sample_contact_zip/postal_code	53705
Sample_contact_country	USA
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM1154nnn/GSM1154503/suppl/GSM1154503_norm_7_9_left_RG230_2.0_110707.CEL.gz
Sample_series_id	GSE47676
Sample_data_row_count	31099

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