Result

Search results for the GEO ID: GSE17745

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Description

Characteristics

GSM442987

GPL1261

BM_mutant_rep1

bone marrow macrophages

strain: c57BL/6 cell type: TNFR1-/-R2-/- BMMs expressing a chimeric receptor consisting of the external domain of mouse TNFR1 linked to the transmembrane and intracellular domain of mouse RANK bearing inactivating mutations in the IVVY motif

none

Sample_geo_accession	GSM442987
Sample_status	Public on Aug 20 2011
Sample_submission_date	Aug 20 2009
Sample_last_update_date	Aug 20 2011
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_biomaterial_provider_ch1	Xu Feng, UAB
Sample_treatment_protocol_ch1	TNFR1-/-R2-/- BMMs expressing a chimeric receptor consisting of the external domain of mouse TNFR1 linked to the transmembrane and intracellular domain of mouse RANK (WT) or TNFR1-/-R2-/- BMMs expressing a chimeric receptor consisting of the external domain of mouse TNFR1 linked to the transmembrane and intracellular domain of mouse RANK bearing inactivating mutations in the IVVY motif (Mu) were cultured in six 60-mm tissue culture dishes with α-MEM containing 10% heat-inactivated FBS and treated with M-CSF (44ng/ml) and TNFα (10ng/ml) for 24 hours. Total RNA was isolated from the six dishes and pooled. The RNA sample preparation was repeated independently two more times. Three sets of total RNA samples prepared from three independent assays were subject to microarray analysis using Mouse Genome 430 2.0 Array at the UAB Microarray Shared Facility.
Sample_growth_protocol_ch1	TNFR1-/-R2-/- BMMs were isolated from TNFR1-/-R2-/- double KO mice from The Jackson Laboratory (Bar Harbor, ME). TNFR1-/-R2-/- BMMs expressing WT chimeric receptor or Mu chimeric receptor were prepared by infecting TNFR1-/-R2-/- BMMs with retrovirus encoding WT chimeric receptor or Mu chimeric receptor. Positive cells were selected with puromycin (2ug/ml) for 3 days.
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Standard Affymetrix protocol
Sample_label_ch1	Biotin
Sample_label_protocol_ch1	The labeling of the RNA was done as per the manufacturers instructions (Affymetrix, Santa Clara, CA). Briefly, One microgram of total RNA was reverse transcribed using an oligo dT-T7 primer under standard conditions. The resulting first strand cDNA was incubated with RNAse H, E. coli DNA ligase, and E. coli DNA polymerase, dNTPs and buffer to produce second-stranded cDNA. The cDNA was purified using spin columns following the manufacturer’s instructions (Qiagen). The purified cDNA was then used in an in vitro transcription assay to produce the target biotin labeled cRNA for hybridization using the 3’ IVT kit from Affymetrix following the manufacturer’s instructions. The cRNA was purified using a spin column following the kit’s instructions (Qiagen), and fragmented prior to hybridization onto the GeneChips.
Sample_hyb_protocol	Hybridization of the GeneChips was carried out in the following the manufacturer’s protocol. Briefly, the fragmented biotin labeled cRNA was mixed with 0.05nM control oligonucleotide B2, 20X Eukaryotic hybridization controls (bioB, bioC, bioD, cre), 0.1 mg/mL herring sperm DNA, 0.5 mg/mL BSA, 2X hybridization buffer (1X 100mM MES/1M NaCl/20mM EDTA/0.01% Tween-20) and 10% DMSO. The hybridization cocktail was incubated at 99oC for 5 minutes to denature the herring sperm DNA and any secondary structure on the cRNA. The cocktail was then equilibrated to 45oC for 5 minutes prior to addition to the GeneChip. Hybridiztion was carried out in the Affymetrix Hybridization Oven 640 at 45oC with rotation at 60rpm for 16 hours. Following the hybridization step the GeneChips were washed and stained in the Affymetrix Fluidics Station 450 following Fluidics Protocol Mini_euk2v3 as recommended by the manufacturer for this Chip type. The GeneChips were first washed in a non-stringent wash buffer of 6X SSPE/0.01% Tween-20 followed by stringent wash with 100mM MES/0.1M NaCl/0.01% Tween-20. The first staining of the GeneChips was done in 2X stain buffer (1X 100mM MES/1M NaCl/0.05% Tween-20), 2mg/mL BSA, and 0.01 mg/mL Streptavidin Phycoerythrin (SAPE). The GeneChips were washed in non-stringent wash buffer. A second stain comprised of 2X staining buffer, 2mg/mL BSA, 0.1mg/mL Goat IgG, and 0.003mg/mL of biotinylated antibody was conducted immediately followed by a third stain comprised of the SAPE solution (see above). Finally, the GeneChips were washed in non-stringent wash buffer prior to scanning.
Sample_scan_protocol	Scanning of the GeneChips was done using the Affymetrix 3000 7G scanner as described by the manufacturer. The Fluidics station and Scanner were controlled using Affymetrix GeneChip Command Console (AGCC).
Sample_data_processing	The raw data were obtained through GCOS, background subtracted, and normalized through GC-RMA methods.
Sample_platform_id	GPL1261
Sample_contact_name	Dongquan,,Chen
Sample_contact_email	dongquan@uab.edu
Sample_contact_phone	2059757131
Sample_contact_laboratory	Preventive Medicine
Sample_contact_department	Medicine
Sample_contact_institute	Univ of Alabama at Birmingham
Sample_contact_address	1717 11th Ave South
Sample_contact_city	Birmingham
Sample_contact_state	AL
Sample_contact_zip/postal_code	35294
Sample_contact_country	USA
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM442nnn/GSM442987/suppl/GSM442987_mu1.CEL.gz
Sample_series_id	GSE17745
Sample_data_row_count	45101

GSM442988

GPL1261

BM_mutant_rep2

bone marrow macrophages

none

Sample_geo_accession	GSM442988
Sample_status	Public on Aug 20 2011
Sample_submission_date	Aug 20 2009
Sample_last_update_date	Aug 20 2011
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_biomaterial_provider_ch1	Xu Feng, UAB
Sample_treatment_protocol_ch1	TNFR1-/-R2-/- BMMs expressing a chimeric receptor consisting of the external domain of mouse TNFR1 linked to the transmembrane and intracellular domain of mouse RANK (WT) or TNFR1-/-R2-/- BMMs expressing a chimeric receptor consisting of the external domain of mouse TNFR1 linked to the transmembrane and intracellular domain of mouse RANK bearing inactivating mutations in the IVVY motif (Mu) were cultured in six 60-mm tissue culture dishes with α-MEM containing 10% heat-inactivated FBS and treated with M-CSF (44ng/ml) and TNFα (10ng/ml) for 24 hours. Total RNA was isolated from the six dishes and pooled. The RNA sample preparation was repeated independently two more times. Three sets of total RNA samples prepared from three independent assays were subject to microarray analysis using Mouse Genome 430 2.0 Array at the UAB Microarray Shared Facility.
Sample_growth_protocol_ch1	TNFR1-/-R2-/- BMMs were isolated from TNFR1-/-R2-/- double KO mice from The Jackson Laboratory (Bar Harbor, ME). TNFR1-/-R2-/- BMMs expressing WT chimeric receptor or Mu chimeric receptor were prepared by infecting TNFR1-/-R2-/- BMMs with retrovirus encoding WT chimeric receptor or Mu chimeric receptor. Positive cells were selected with puromycin (2ug/ml) for 3 days.
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Standard Affymetrix protocol
Sample_label_ch1	Biotin
Sample_label_protocol_ch1	The labeling of the RNA was done as per the manufacturers instructions (Affymetrix, Santa Clara, CA). Briefly, One microgram of total RNA was reverse transcribed using an oligo dT-T7 primer under standard conditions. The resulting first strand cDNA was incubated with RNAse H, E. coli DNA ligase, and E. coli DNA polymerase, dNTPs and buffer to produce second-stranded cDNA. The cDNA was purified using spin columns following the manufacturer’s instructions (Qiagen). The purified cDNA was then used in an in vitro transcription assay to produce the target biotin labeled cRNA for hybridization using the 3’ IVT kit from Affymetrix following the manufacturer’s instructions. The cRNA was purified using a spin column following the kit’s instructions (Qiagen), and fragmented prior to hybridization onto the GeneChips.
Sample_hyb_protocol	Hybridization of the GeneChips was carried out in the following the manufacturer’s protocol. Briefly, the fragmented biotin labeled cRNA was mixed with 0.05nM control oligonucleotide B2, 20X Eukaryotic hybridization controls (bioB, bioC, bioD, cre), 0.1 mg/mL herring sperm DNA, 0.5 mg/mL BSA, 2X hybridization buffer (1X 100mM MES/1M NaCl/20mM EDTA/0.01% Tween-20) and 10% DMSO. The hybridization cocktail was incubated at 99oC for 5 minutes to denature the herring sperm DNA and any secondary structure on the cRNA. The cocktail was then equilibrated to 45oC for 5 minutes prior to addition to the GeneChip. Hybridiztion was carried out in the Affymetrix Hybridization Oven 640 at 45oC with rotation at 60rpm for 16 hours. Following the hybridization step the GeneChips were washed and stained in the Affymetrix Fluidics Station 450 following Fluidics Protocol Mini_euk2v3 as recommended by the manufacturer for this Chip type. The GeneChips were first washed in a non-stringent wash buffer of 6X SSPE/0.01% Tween-20 followed by stringent wash with 100mM MES/0.1M NaCl/0.01% Tween-20. The first staining of the GeneChips was done in 2X stain buffer (1X 100mM MES/1M NaCl/0.05% Tween-20), 2mg/mL BSA, and 0.01 mg/mL Streptavidin Phycoerythrin (SAPE). The GeneChips were washed in non-stringent wash buffer. A second stain comprised of 2X staining buffer, 2mg/mL BSA, 0.1mg/mL Goat IgG, and 0.003mg/mL of biotinylated antibody was conducted immediately followed by a third stain comprised of the SAPE solution (see above). Finally, the GeneChips were washed in non-stringent wash buffer prior to scanning.
Sample_scan_protocol	Scanning of the GeneChips was done using the Affymetrix 3000 7G scanner as described by the manufacturer. The Fluidics station and Scanner were controlled using Affymetrix GeneChip Command Console (AGCC).
Sample_data_processing	The raw data were obtained through GCOS, background subtracted, and normalized through GC-RMA methods.
Sample_platform_id	GPL1261
Sample_contact_name	Dongquan,,Chen
Sample_contact_email	dongquan@uab.edu
Sample_contact_phone	2059757131
Sample_contact_laboratory	Preventive Medicine
Sample_contact_department	Medicine
Sample_contact_institute	Univ of Alabama at Birmingham
Sample_contact_address	1717 11th Ave South
Sample_contact_city	Birmingham
Sample_contact_state	AL
Sample_contact_zip/postal_code	35294
Sample_contact_country	USA
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM442nnn/GSM442988/suppl/GSM442988_mu2.CEL.gz
Sample_series_id	GSE17745
Sample_data_row_count	45101

GSM442989

GPL1261

BM_mutant_rep3

bone marrow macrophages

none

Sample_geo_accession	GSM442989
Sample_status	Public on Aug 20 2011
Sample_submission_date	Aug 20 2009
Sample_last_update_date	Aug 20 2011
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_biomaterial_provider_ch1	Xu Feng, UAB
Sample_treatment_protocol_ch1	TNFR1-/-R2-/- BMMs expressing a chimeric receptor consisting of the external domain of mouse TNFR1 linked to the transmembrane and intracellular domain of mouse RANK (WT) or TNFR1-/-R2-/- BMMs expressing a chimeric receptor consisting of the external domain of mouse TNFR1 linked to the transmembrane and intracellular domain of mouse RANK bearing inactivating mutations in the IVVY motif (Mu) were cultured in six 60-mm tissue culture dishes with α-MEM containing 10% heat-inactivated FBS and treated with M-CSF (44ng/ml) and TNFα (10ng/ml) for 24 hours. Total RNA was isolated from the six dishes and pooled. The RNA sample preparation was repeated independently two more times. Three sets of total RNA samples prepared from three independent assays were subject to microarray analysis using Mouse Genome 430 2.0 Array at the UAB Microarray Shared Facility.
Sample_growth_protocol_ch1	TNFR1-/-R2-/- BMMs were isolated from TNFR1-/-R2-/- double KO mice from The Jackson Laboratory (Bar Harbor, ME). TNFR1-/-R2-/- BMMs expressing WT chimeric receptor or Mu chimeric receptor were prepared by infecting TNFR1-/-R2-/- BMMs with retrovirus encoding WT chimeric receptor or Mu chimeric receptor. Positive cells were selected with puromycin (2ug/ml) for 3 days.
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Standard Affymetrix protocol
Sample_label_ch1	Biotin
Sample_label_protocol_ch1	The labeling of the RNA was done as per the manufacturers instructions (Affymetrix, Santa Clara, CA). Briefly, One microgram of total RNA was reverse transcribed using an oligo dT-T7 primer under standard conditions. The resulting first strand cDNA was incubated with RNAse H, E. coli DNA ligase, and E. coli DNA polymerase, dNTPs and buffer to produce second-stranded cDNA. The cDNA was purified using spin columns following the manufacturer’s instructions (Qiagen). The purified cDNA was then used in an in vitro transcription assay to produce the target biotin labeled cRNA for hybridization using the 3’ IVT kit from Affymetrix following the manufacturer’s instructions. The cRNA was purified using a spin column following the kit’s instructions (Qiagen), and fragmented prior to hybridization onto the GeneChips.
Sample_hyb_protocol	Hybridization of the GeneChips was carried out in the following the manufacturer’s protocol. Briefly, the fragmented biotin labeled cRNA was mixed with 0.05nM control oligonucleotide B2, 20X Eukaryotic hybridization controls (bioB, bioC, bioD, cre), 0.1 mg/mL herring sperm DNA, 0.5 mg/mL BSA, 2X hybridization buffer (1X 100mM MES/1M NaCl/20mM EDTA/0.01% Tween-20) and 10% DMSO. The hybridization cocktail was incubated at 99oC for 5 minutes to denature the herring sperm DNA and any secondary structure on the cRNA. The cocktail was then equilibrated to 45oC for 5 minutes prior to addition to the GeneChip. Hybridiztion was carried out in the Affymetrix Hybridization Oven 640 at 45oC with rotation at 60rpm for 16 hours. Following the hybridization step the GeneChips were washed and stained in the Affymetrix Fluidics Station 450 following Fluidics Protocol Mini_euk2v3 as recommended by the manufacturer for this Chip type. The GeneChips were first washed in a non-stringent wash buffer of 6X SSPE/0.01% Tween-20 followed by stringent wash with 100mM MES/0.1M NaCl/0.01% Tween-20. The first staining of the GeneChips was done in 2X stain buffer (1X 100mM MES/1M NaCl/0.05% Tween-20), 2mg/mL BSA, and 0.01 mg/mL Streptavidin Phycoerythrin (SAPE). The GeneChips were washed in non-stringent wash buffer. A second stain comprised of 2X staining buffer, 2mg/mL BSA, 0.1mg/mL Goat IgG, and 0.003mg/mL of biotinylated antibody was conducted immediately followed by a third stain comprised of the SAPE solution (see above). Finally, the GeneChips were washed in non-stringent wash buffer prior to scanning.
Sample_scan_protocol	Scanning of the GeneChips was done using the Affymetrix 3000 7G scanner as described by the manufacturer. The Fluidics station and Scanner were controlled using Affymetrix GeneChip Command Console (AGCC).
Sample_data_processing	The raw data were obtained through GCOS, background subtracted, and normalized through GC-RMA methods.
Sample_platform_id	GPL1261
Sample_contact_name	Dongquan,,Chen
Sample_contact_email	dongquan@uab.edu
Sample_contact_phone	2059757131
Sample_contact_laboratory	Preventive Medicine
Sample_contact_department	Medicine
Sample_contact_institute	Univ of Alabama at Birmingham
Sample_contact_address	1717 11th Ave South
Sample_contact_city	Birmingham
Sample_contact_state	AL
Sample_contact_zip/postal_code	35294
Sample_contact_country	USA
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM442nnn/GSM442989/suppl/GSM442989_mu3.CEL.gz
Sample_series_id	GSE17745
Sample_data_row_count	45101

GSM442990

GPL1261

BM_wt_rep1

bone marrow macrophages

strain: c57BL/6 cell type: TNFR1-/-R2-/- BMMs expressing a chimeric receptor consisting of the external domain of mouse TNFR1 linked to the transmembrane and intracellular domain of mouse RANK

none

Sample_geo_accession	GSM442990
Sample_status	Public on Aug 20 2011
Sample_submission_date	Aug 20 2009
Sample_last_update_date	Aug 20 2011
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_biomaterial_provider_ch1	Xu Feng, UAB
Sample_treatment_protocol_ch1	TNFR1-/-R2-/- BMMs expressing a chimeric receptor consisting of the external domain of mouse TNFR1 linked to the transmembrane and intracellular domain of mouse RANK (WT) or TNFR1-/-R2-/- BMMs expressing a chimeric receptor consisting of the external domain of mouse TNFR1 linked to the transmembrane and intracellular domain of mouse RANK bearing inactivating mutations in the IVVY motif (Mu) were cultured in six 60-mm tissue culture dishes with α-MEM containing 10% heat-inactivated FBS and treated with M-CSF (44ng/ml) and TNFα (10ng/ml) for 24 hours. Total RNA was isolated from the six dishes and pooled. The RNA sample preparation was repeated independently two more times. Three sets of total RNA samples prepared from three independent assays were subject to microarray analysis using Mouse Genome 430 2.0 Array at the UAB Microarray Shared Facility.
Sample_growth_protocol_ch1	TNFR1-/-R2-/- BMMs were isolated from TNFR1-/-R2-/- double KO mice from The Jackson Laboratory (Bar Harbor, ME). TNFR1-/-R2-/- BMMs expressing WT chimeric receptor or Mu chimeric receptor were prepared by infecting TNFR1-/-R2-/- BMMs with retrovirus encoding WT chimeric receptor or Mu chimeric receptor. Positive cells were selected with puromycin (2ug/ml) for 3 days.
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Standard Affymetrix protocol
Sample_label_ch1	Biotin
Sample_label_protocol_ch1	The labeling of the RNA was done as per the manufacturers instructions (Affymetrix, Santa Clara, CA). Briefly, One microgram of total RNA was reverse transcribed using an oligo dT-T7 primer under standard conditions. The resulting first strand cDNA was incubated with RNAse H, E. coli DNA ligase, and E. coli DNA polymerase, dNTPs and buffer to produce second-stranded cDNA. The cDNA was purified using spin columns following the manufacturer’s instructions (Qiagen). The purified cDNA was then used in an in vitro transcription assay to produce the target biotin labeled cRNA for hybridization using the 3’ IVT kit from Affymetrix following the manufacturer’s instructions. The cRNA was purified using a spin column following the kit’s instructions (Qiagen), and fragmented prior to hybridization onto the GeneChips.
Sample_hyb_protocol	Hybridization of the GeneChips was carried out in the following the manufacturer’s protocol. Briefly, the fragmented biotin labeled cRNA was mixed with 0.05nM control oligonucleotide B2, 20X Eukaryotic hybridization controls (bioB, bioC, bioD, cre), 0.1 mg/mL herring sperm DNA, 0.5 mg/mL BSA, 2X hybridization buffer (1X 100mM MES/1M NaCl/20mM EDTA/0.01% Tween-20) and 10% DMSO. The hybridization cocktail was incubated at 99oC for 5 minutes to denature the herring sperm DNA and any secondary structure on the cRNA. The cocktail was then equilibrated to 45oC for 5 minutes prior to addition to the GeneChip. Hybridiztion was carried out in the Affymetrix Hybridization Oven 640 at 45oC with rotation at 60rpm for 16 hours. Following the hybridization step the GeneChips were washed and stained in the Affymetrix Fluidics Station 450 following Fluidics Protocol Mini_euk2v3 as recommended by the manufacturer for this Chip type. The GeneChips were first washed in a non-stringent wash buffer of 6X SSPE/0.01% Tween-20 followed by stringent wash with 100mM MES/0.1M NaCl/0.01% Tween-20. The first staining of the GeneChips was done in 2X stain buffer (1X 100mM MES/1M NaCl/0.05% Tween-20), 2mg/mL BSA, and 0.01 mg/mL Streptavidin Phycoerythrin (SAPE). The GeneChips were washed in non-stringent wash buffer. A second stain comprised of 2X staining buffer, 2mg/mL BSA, 0.1mg/mL Goat IgG, and 0.003mg/mL of biotinylated antibody was conducted immediately followed by a third stain comprised of the SAPE solution (see above). Finally, the GeneChips were washed in non-stringent wash buffer prior to scanning.
Sample_scan_protocol	Scanning of the GeneChips was done using the Affymetrix 3000 7G scanner as described by the manufacturer. The Fluidics station and Scanner were controlled using Affymetrix GeneChip Command Console (AGCC).
Sample_data_processing	The raw data were obtained through GCOS, background subtracted, and normalized through GC-RMA methods.
Sample_platform_id	GPL1261
Sample_contact_name	Dongquan,,Chen
Sample_contact_email	dongquan@uab.edu
Sample_contact_phone	2059757131
Sample_contact_laboratory	Preventive Medicine
Sample_contact_department	Medicine
Sample_contact_institute	Univ of Alabama at Birmingham
Sample_contact_address	1717 11th Ave South
Sample_contact_city	Birmingham
Sample_contact_state	AL
Sample_contact_zip/postal_code	35294
Sample_contact_country	USA
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM442nnn/GSM442990/suppl/GSM442990_wt1.CEL.gz
Sample_series_id	GSE17745
Sample_data_row_count	45101

GSM442991

GPL1261

BM_wt_rep2

bone marrow macrophages

strain: c57BL/6 cell type: TNFR1-/-R2-/- BMMs expressing a chimeric receptor consisting of the external domain of mouse TNFR1 linked to the transmembrane and intracellular domain of mouse RANK

none

Sample_geo_accession	GSM442991
Sample_status	Public on Aug 20 2011
Sample_submission_date	Aug 20 2009
Sample_last_update_date	Aug 20 2011
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_biomaterial_provider_ch1	Xu Feng, UAB
Sample_treatment_protocol_ch1	TNFR1-/-R2-/- BMMs expressing a chimeric receptor consisting of the external domain of mouse TNFR1 linked to the transmembrane and intracellular domain of mouse RANK (WT) or TNFR1-/-R2-/- BMMs expressing a chimeric receptor consisting of the external domain of mouse TNFR1 linked to the transmembrane and intracellular domain of mouse RANK bearing inactivating mutations in the IVVY motif (Mu) were cultured in six 60-mm tissue culture dishes with α-MEM containing 10% heat-inactivated FBS and treated with M-CSF (44ng/ml) and TNFα (10ng/ml) for 24 hours. Total RNA was isolated from the six dishes and pooled. The RNA sample preparation was repeated independently two more times. Three sets of total RNA samples prepared from three independent assays were subject to microarray analysis using Mouse Genome 430 2.0 Array at the UAB Microarray Shared Facility.
Sample_growth_protocol_ch1	TNFR1-/-R2-/- BMMs were isolated from TNFR1-/-R2-/- double KO mice from The Jackson Laboratory (Bar Harbor, ME). TNFR1-/-R2-/- BMMs expressing WT chimeric receptor or Mu chimeric receptor were prepared by infecting TNFR1-/-R2-/- BMMs with retrovirus encoding WT chimeric receptor or Mu chimeric receptor. Positive cells were selected with puromycin (2ug/ml) for 3 days.
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Standard Affymetrix protocol
Sample_label_ch1	Biotin
Sample_label_protocol_ch1	The labeling of the RNA was done as per the manufacturers instructions (Affymetrix, Santa Clara, CA). Briefly, One microgram of total RNA was reverse transcribed using an oligo dT-T7 primer under standard conditions. The resulting first strand cDNA was incubated with RNAse H, E. coli DNA ligase, and E. coli DNA polymerase, dNTPs and buffer to produce second-stranded cDNA. The cDNA was purified using spin columns following the manufacturer’s instructions (Qiagen). The purified cDNA was then used in an in vitro transcription assay to produce the target biotin labeled cRNA for hybridization using the 3’ IVT kit from Affymetrix following the manufacturer’s instructions. The cRNA was purified using a spin column following the kit’s instructions (Qiagen), and fragmented prior to hybridization onto the GeneChips.
Sample_hyb_protocol	Hybridization of the GeneChips was carried out in the following the manufacturer’s protocol. Briefly, the fragmented biotin labeled cRNA was mixed with 0.05nM control oligonucleotide B2, 20X Eukaryotic hybridization controls (bioB, bioC, bioD, cre), 0.1 mg/mL herring sperm DNA, 0.5 mg/mL BSA, 2X hybridization buffer (1X 100mM MES/1M NaCl/20mM EDTA/0.01% Tween-20) and 10% DMSO. The hybridization cocktail was incubated at 99oC for 5 minutes to denature the herring sperm DNA and any secondary structure on the cRNA. The cocktail was then equilibrated to 45oC for 5 minutes prior to addition to the GeneChip. Hybridiztion was carried out in the Affymetrix Hybridization Oven 640 at 45oC with rotation at 60rpm for 16 hours. Following the hybridization step the GeneChips were washed and stained in the Affymetrix Fluidics Station 450 following Fluidics Protocol Mini_euk2v3 as recommended by the manufacturer for this Chip type. The GeneChips were first washed in a non-stringent wash buffer of 6X SSPE/0.01% Tween-20 followed by stringent wash with 100mM MES/0.1M NaCl/0.01% Tween-20. The first staining of the GeneChips was done in 2X stain buffer (1X 100mM MES/1M NaCl/0.05% Tween-20), 2mg/mL BSA, and 0.01 mg/mL Streptavidin Phycoerythrin (SAPE). The GeneChips were washed in non-stringent wash buffer. A second stain comprised of 2X staining buffer, 2mg/mL BSA, 0.1mg/mL Goat IgG, and 0.003mg/mL of biotinylated antibody was conducted immediately followed by a third stain comprised of the SAPE solution (see above). Finally, the GeneChips were washed in non-stringent wash buffer prior to scanning.
Sample_scan_protocol	Scanning of the GeneChips was done using the Affymetrix 3000 7G scanner as described by the manufacturer. The Fluidics station and Scanner were controlled using Affymetrix GeneChip Command Console (AGCC).
Sample_data_processing	The raw data were obtained through GCOS, background subtracted, and normalized through GC-RMA methods.
Sample_platform_id	GPL1261
Sample_contact_name	Dongquan,,Chen
Sample_contact_email	dongquan@uab.edu
Sample_contact_phone	2059757131
Sample_contact_laboratory	Preventive Medicine
Sample_contact_department	Medicine
Sample_contact_institute	Univ of Alabama at Birmingham
Sample_contact_address	1717 11th Ave South
Sample_contact_city	Birmingham
Sample_contact_state	AL
Sample_contact_zip/postal_code	35294
Sample_contact_country	USA
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM442nnn/GSM442991/suppl/GSM442991_wt2.cel.gz
Sample_series_id	GSE17745
Sample_data_row_count	45101

GSM442992

GPL1261

BM_wt_rep3

bone marrow macrophages

strain: c57BL/6 cell type: TNFR1-/-R2-/- BMMs expressing a chimeric receptor consisting of the external domain of mouse TNFR1 linked to the transmembrane and intracellular domain of mouse RANK

none

Sample_geo_accession	GSM442992
Sample_status	Public on Aug 20 2011
Sample_submission_date	Aug 20 2009
Sample_last_update_date	Aug 20 2011
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_biomaterial_provider_ch1	Xu Feng, UAB
Sample_treatment_protocol_ch1	TNFR1-/-R2-/- BMMs expressing a chimeric receptor consisting of the external domain of mouse TNFR1 linked to the transmembrane and intracellular domain of mouse RANK (WT) or TNFR1-/-R2-/- BMMs expressing a chimeric receptor consisting of the external domain of mouse TNFR1 linked to the transmembrane and intracellular domain of mouse RANK bearing inactivating mutations in the IVVY motif (Mu) were cultured in six 60-mm tissue culture dishes with α-MEM containing 10% heat-inactivated FBS and treated with M-CSF (44ng/ml) and TNFα (10ng/ml) for 24 hours. Total RNA was isolated from the six dishes and pooled. The RNA sample preparation was repeated independently two more times. Three sets of total RNA samples prepared from three independent assays were subject to microarray analysis using Mouse Genome 430 2.0 Array at the UAB Microarray Shared Facility.
Sample_growth_protocol_ch1	TNFR1-/-R2-/- BMMs were isolated from TNFR1-/-R2-/- double KO mice from The Jackson Laboratory (Bar Harbor, ME). TNFR1-/-R2-/- BMMs expressing WT chimeric receptor or Mu chimeric receptor were prepared by infecting TNFR1-/-R2-/- BMMs with retrovirus encoding WT chimeric receptor or Mu chimeric receptor. Positive cells were selected with puromycin (2ug/ml) for 3 days.
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Standard Affymetrix protocol
Sample_label_ch1	Biotin
Sample_label_protocol_ch1	The labeling of the RNA was done as per the manufacturers instructions (Affymetrix, Santa Clara, CA). Briefly, One microgram of total RNA was reverse transcribed using an oligo dT-T7 primer under standard conditions. The resulting first strand cDNA was incubated with RNAse H, E. coli DNA ligase, and E. coli DNA polymerase, dNTPs and buffer to produce second-stranded cDNA. The cDNA was purified using spin columns following the manufacturer’s instructions (Qiagen). The purified cDNA was then used in an in vitro transcription assay to produce the target biotin labeled cRNA for hybridization using the 3’ IVT kit from Affymetrix following the manufacturer’s instructions. The cRNA was purified using a spin column following the kit’s instructions (Qiagen), and fragmented prior to hybridization onto the GeneChips.
Sample_hyb_protocol	Hybridization of the GeneChips was carried out in the following the manufacturer’s protocol. Briefly, the fragmented biotin labeled cRNA was mixed with 0.05nM control oligonucleotide B2, 20X Eukaryotic hybridization controls (bioB, bioC, bioD, cre), 0.1 mg/mL herring sperm DNA, 0.5 mg/mL BSA, 2X hybridization buffer (1X 100mM MES/1M NaCl/20mM EDTA/0.01% Tween-20) and 10% DMSO. The hybridization cocktail was incubated at 99oC for 5 minutes to denature the herring sperm DNA and any secondary structure on the cRNA. The cocktail was then equilibrated to 45oC for 5 minutes prior to addition to the GeneChip. Hybridiztion was carried out in the Affymetrix Hybridization Oven 640 at 45oC with rotation at 60rpm for 16 hours. Following the hybridization step the GeneChips were washed and stained in the Affymetrix Fluidics Station 450 following Fluidics Protocol Mini_euk2v3 as recommended by the manufacturer for this Chip type. The GeneChips were first washed in a non-stringent wash buffer of 6X SSPE/0.01% Tween-20 followed by stringent wash with 100mM MES/0.1M NaCl/0.01% Tween-20. The first staining of the GeneChips was done in 2X stain buffer (1X 100mM MES/1M NaCl/0.05% Tween-20), 2mg/mL BSA, and 0.01 mg/mL Streptavidin Phycoerythrin (SAPE). The GeneChips were washed in non-stringent wash buffer. A second stain comprised of 2X staining buffer, 2mg/mL BSA, 0.1mg/mL Goat IgG, and 0.003mg/mL of biotinylated antibody was conducted immediately followed by a third stain comprised of the SAPE solution (see above). Finally, the GeneChips were washed in non-stringent wash buffer prior to scanning.
Sample_scan_protocol	Scanning of the GeneChips was done using the Affymetrix 3000 7G scanner as described by the manufacturer. The Fluidics station and Scanner were controlled using Affymetrix GeneChip Command Console (AGCC).
Sample_data_processing	The raw data were obtained through GCOS, background subtracted, and normalized through GC-RMA methods.
Sample_platform_id	GPL1261
Sample_contact_name	Dongquan,,Chen
Sample_contact_email	dongquan@uab.edu
Sample_contact_phone	2059757131
Sample_contact_laboratory	Preventive Medicine
Sample_contact_department	Medicine
Sample_contact_institute	Univ of Alabama at Birmingham
Sample_contact_address	1717 11th Ave South
Sample_contact_city	Birmingham
Sample_contact_state	AL
Sample_contact_zip/postal_code	35294
Sample_contact_country	USA
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM442nnn/GSM442992/suppl/GSM442992_wt3.CEL.gz
Sample_series_id	GSE17745
Sample_data_row_count	45101

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