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Search results for the GEO ID: GSE15733

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GSM393497

GPL1261

CD44highCD62L-CD25- CD4+ T cells from C57BL/6 murine bone marrow_rep1

CD44highCD62L-CD25- CD4+ T cells from C57BL/6 murine bone marrow

cell type: CD44highCD62L-CD25- CD4+ T cells tissue: bone marrow

FACSAria sorted CD44highCD62L-CD25- CD4+ T cells of murine (C57BL/6 mice) bone marrow were compared to those of the spleen using Affymetrix GeneChip Mouse Genome 430A 2.0 Array. After total RNA extraction, reverse transcription, cDNA extraction, the biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized in triplicates for each of the two groups to the GeneChip arrays. Group of bone marrow chips: BMCD4T1, BMCD4T2, BMCD4T3. Group of spleen chips: SCD4T1, SCD4T2, SCD4T3.

Sample_geo_accession	GSM393497
Sample_status	Public on Apr 21 2009
Sample_submission_date	Apr 17 2009
Sample_last_update_date	Apr 20 2009
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_biomaterial_provider_ch1	C57BL/6 mice from DRFZ
Sample_treatment_protocol_ch1	CD44highCD62L-CD25- CD4+ T cells from spleen or bone marrow of 12-16 week old C57BL/6 mice were sorted with FACSAria
Sample_growth_protocol_ch1	n/a
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was extracted using Trizol reagent (Invitrogen). The amount, purity, and integrity of RNA was assessed for each sample using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip and a NanoDrop ND-1000 spectrophotometer. Contaminating genomic DNA was removed by an on-column DNA digestion step (Qiagen). Ten micrograms of total RNA was reverse-transcribed, followed by cDNA extraction using a PhaseLock gel (Eppendorf), and precipitation with ethanol and ammonium acetate.
Sample_label_ch1	biotin
Sample_label_protocol_ch1	10 µg of total RNA from each cell sample was reverse transcribed using T7-(d)T24 primer and SuperScript II reverse transcriptase, following cDNA extraction with a PhaseLock gel (Eppendorf), and precipitation with ethanol and ammonium acetate. Blocking was according to manufactures recommendations and probes were subsequently hybridized. After washing the hybridization signals were visualized by staining with streptavidin-phycoerythrin and amplification with anti-streptavidin.
Sample_hyb_protocol	Biotinylated cRNA was in vitro transcribed using the MEGAscript high yield transcription kit (Ambion) according to the manufacturer's recommendations. Biotinylated cRNA was fragmented, and the hybridization cocktail was prepared according to Affymetrix protocols (15 µg fragmented biotin-labeled cRNA spiked with Eukaryotic hybridization controls). The Murine Genome 430A version 2 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400.
Sample_scan_protocol	Arrays were scanned on an Affymetrix GeneChip Scanner 3000
Sample_data_processing	Data were analyzed according to High Performance Chip Data Analysis (HPCDA, unpublished - Joachim R. Gruen) with the Bioretis database (http://www.bioretis-analysis.de/) using the default filter parameters for decreased and increased gene lists (description of database c.f. open access paper Menssen et al., 2009; PMID: 19265543). Chip data included in the Bioretis database were analyzed using the GeneChip Operating Software (GCOS, Affymetrix), version 1.4. Microarrays were globally normalized and scaled to a trimmed mean expression value of 150. Quality checks were performed according to the manufacturer's recommendations. All 3 + 3 chips were compared to each other (bone marrow group vs. spleen, 9 comparisons, and all chips within the groups, twice 6 comparisons), and the following parameters of absolute and comparative analysis were included in the Bioretis database: expression heights (Signals) and mean, median and standard deviation of Signals of both groups, call for presence of transcripts (Absent, A; Marginal, M; Present, P), p value for presence or absence of transcripts, log2 value of fold change (Signal Log Ratio, SLR) and the calculated fold change as mean values, call for the significance of differentially expression (Change Call: Increase, I; Marginal I, MI; Decrease, D; Marginal D, MD; No Change, NC), and the p value for that call. Additionally – not with GCOS calculated – t tests of log Signals and SLRs were included in the database. For each present transcript the significance of differential expression between the groups of arrays was either calculated using strict Bonferroni corrected Welch t tests between 9 SLR values of bone marrow vs. spleen and 12 SLR values within both groups (the latter 12 giving a mean SLR value of zero; p-value had to be <= 5.279E-08) or more than 50% of non parametrically calculated Change calls (Mann-Whitney U test, GCOS) i.e. 5 or more of all 9 Change calls have to be in the same direction. Significantly differentially expressed genes were filtered using the both default parameter sets of filter criteria; these are a combination of four different queries. Filter criteria were developed with various data sets of GeneChips and validated with the Affymetrix Latin Square dataset as shown in Menssen et al., 2009; PMID: 19265543.
Sample_platform_id	GPL1261
Sample_contact_name	Joachim,R.,Grün
Sample_contact_email	Gruen@DRFZ.de
Sample_contact_department	Bioinformatics
Sample_contact_institute	Deutsches Rheuma-Forschungszentrum (DRFZ)
Sample_contact_address	Charitéplatz 1
Sample_contact_city	Berlin
Sample_contact_zip/postal_code	D-10117
Sample_contact_country	Germany
Sample_contact_web_link	www.drfz.de
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM393nnn/GSM393497/suppl/GSM393497.CEL.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM393nnn/GSM393497/suppl/GSM393497.CHP.gz
Sample_series_id	GSE15733
Sample_data_row_count	45101

GSM393940

GPL1261

CD44highCD62L-CD25- CD4+ T cells from C57BL/6 murine bone marrow_rep2

CD44highCD62L-CD25- CD4+ T cells from C57BL/6 murine bone marrow

cell type: CD44highCD62L-CD25- CD4+ T cells tissue: bone marrow

Sample_geo_accession	GSM393940
Sample_status	Public on Apr 21 2009
Sample_submission_date	Apr 18 2009
Sample_last_update_date	Apr 20 2009
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_biomaterial_provider_ch1	C57BL/6 mice from DRFZ
Sample_treatment_protocol_ch1	CD44highCD62L-CD25- CD4+ T cells from spleen or bone marrow of 12-16 week old C57BL/6 mice were sorted with FACSAria
Sample_growth_protocol_ch1	n/a
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was extracted using Trizol reagent (Invitrogen). The amount, purity, and integrity of RNA was assessed for each sample using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip and a NanoDrop ND-1000 spectrophotometer. Contaminating genomic DNA was removed by an on-column DNA digestion step (Qiagen). Ten micrograms of total RNA was reverse-transcribed, followed by cDNA extraction using a PhaseLock gel (Eppendorf), and precipitation with ethanol and ammonium acetate.
Sample_label_ch1	biotin
Sample_label_protocol_ch1	10 µg of total RNA from each cell sample was reverse transcribed using T7-(d)T24 primer and SuperScript II reverse transcriptase, following cDNA extraction with a PhaseLock gel (Eppendorf), and precipitation with ethanol and ammonium acetate. Blocking was according to manufactures recommendations and probes were subsequently hybridized. After washing the hybridization signals were visualized by staining with streptavidin-phycoerythrin and amplification with anti-streptavidin.
Sample_hyb_protocol	Biotinylated cRNA was in vitro transcribed using the MEGAscript high yield transcription kit (Ambion) according to the manufacturer's recommendations. Biotinylated cRNA was fragmented, and the hybridization cocktail was prepared according to Affymetrix protocols (15 µg fragmented biotin-labeled cRNA spiked with Eukaryotic hybridization controls). The Murine Genome 430A version 2 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400.
Sample_scan_protocol	Arrays were scanned on an Affymetrix GeneChip Scanner 3000
Sample_data_processing	Data were analyzed according to High Performance Chip Data Analysis (HPCDA, unpublished - Joachim R. Gruen) with the Bioretis database (http://www.bioretis-analysis.de/) using the default filter parameters for decreased and increased gene lists (description of database c.f. open access paper Menssen et al., 2009; PMID: 19265543). Chip data included in the Bioretis database were analyzed using the GeneChip Operating Software (GCOS, Affymetrix), version 1.4. Microarrays were globally normalized and scaled to a trimmed mean expression value of 150. Quality checks were performed according to the manufacturer's recommendations. All 3 + 3 chips were compared to each other (bone marrow group vs. spleen, 9 comparisons, and all chips within the groups, twice 6 comparisons), and the following parameters of absolute and comparative analysis were included in the Bioretis database: expression heights (Signals) and mean, median and standard deviation of Signals of both groups, call for presence of transcripts (Absent, A; Marginal, M; Present, P), p value for presence or absence of transcripts, log2 value of fold change (Signal Log Ratio, SLR) and the calculated fold change as mean values, call for the significance of differentially expression (Change Call: Increase, I; Marginal I, MI; Decrease, D; Marginal D, MD; No Change, NC), and the p value for that call. Additionally – not with GCOS calculated – t tests of log Signals and SLRs were included in the database. For each present transcript the significance of differential expression between the groups of arrays was either calculated using strict Bonferroni corrected Welch t tests between 9 SLR values of bone marrow vs. spleen and 12 SLR values within both groups (the latter 12 giving a mean SLR value of zero; p-value had to be <= 5.279E-08) or more than 50% of non parametrically calculated Change calls (Mann-Whitney U test, GCOS) i.e. 5 or more of all 9 Change calls have to be in the same direction. Significantly differentially expressed genes were filtered using the both default parameter sets of filter criteria; these are a combination of four different queries. Filter criteria were developed with various data sets of GeneChips and validated with the Affymetrix Latin Square dataset as shown in Menssen et al., 2009; PMID: 19265543.
Sample_platform_id	GPL1261
Sample_contact_name	Joachim,R.,Grün
Sample_contact_email	Gruen@DRFZ.de
Sample_contact_department	Bioinformatics
Sample_contact_institute	Deutsches Rheuma-Forschungszentrum (DRFZ)
Sample_contact_address	Charitéplatz 1
Sample_contact_city	Berlin
Sample_contact_zip/postal_code	D-10117
Sample_contact_country	Germany
Sample_contact_web_link	www.drfz.de
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM393nnn/GSM393940/suppl/GSM393940.CEL.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM393nnn/GSM393940/suppl/GSM393940.CHP.gz
Sample_series_id	GSE15733
Sample_data_row_count	45101

GSM393941

GPL1261

CD44highCD62L-CD25- CD4+ T cells from C57BL/6 murine bone marrow_rep3

CD44highCD62L-CD25- CD4+ T cells from C57BL/6 murine bone marrow

cell type: CD44highCD62L-CD25- CD4+ T cells tissue: bone marrow

Sample_geo_accession	GSM393941
Sample_status	Public on Apr 21 2009
Sample_submission_date	Apr 18 2009
Sample_last_update_date	Apr 20 2009
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_biomaterial_provider_ch1	C57BL/6 mice from DRFZ
Sample_treatment_protocol_ch1	CD44highCD62L-CD25- CD4+ T cells from spleen or bone marrow of 12-16 week old C57BL/6 mice were sorted with FACSAria
Sample_growth_protocol_ch1	n/a
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was extracted using Trizol reagent (Invitrogen). The amount, purity, and integrity of RNA was assessed for each sample using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip and a NanoDrop ND-1000 spectrophotometer. Contaminating genomic DNA was removed by an on-column DNA digestion step (Qiagen). Ten micrograms of total RNA was reverse-transcribed, followed by cDNA extraction using a PhaseLock gel (Eppendorf), and precipitation with ethanol and ammonium acetate.
Sample_label_ch1	biotin
Sample_label_protocol_ch1	10 µg of total RNA from each cell sample was reverse transcribed using T7-(d)T24 primer and SuperScript II reverse transcriptase, following cDNA extraction with a PhaseLock gel (Eppendorf), and precipitation with ethanol and ammonium acetate. Blocking was according to manufactures recommendations and probes were subsequently hybridized. After washing the hybridization signals were visualized by staining with streptavidin-phycoerythrin and amplification with anti-streptavidin.
Sample_hyb_protocol	Biotinylated cRNA was in vitro transcribed using the MEGAscript high yield transcription kit (Ambion) according to the manufacturer's recommendations. Biotinylated cRNA was fragmented, and the hybridization cocktail was prepared according to Affymetrix protocols (15 µg fragmented biotin-labeled cRNA spiked with Eukaryotic hybridization controls). The Murine Genome 430A version 2 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400.
Sample_scan_protocol	Arrays were scanned on an Affymetrix GeneChip Scanner 3000
Sample_data_processing	Data were analyzed according to High Performance Chip Data Analysis (HPCDA, unpublished - Joachim R. Gruen) with the Bioretis database (http://www.bioretis-analysis.de/) using the default filter parameters for decreased and increased gene lists (description of database c.f. open access paper Menssen et al., 2009; PMID: 19265543). Chip data included in the Bioretis database were analyzed using the GeneChip Operating Software (GCOS, Affymetrix), version 1.4. Microarrays were globally normalized and scaled to a trimmed mean expression value of 150. Quality checks were performed according to the manufacturer's recommendations. All 3 + 3 chips were compared to each other (bone marrow group vs. spleen, 9 comparisons, and all chips within the groups, twice 6 comparisons), and the following parameters of absolute and comparative analysis were included in the Bioretis database: expression heights (Signals) and mean, median and standard deviation of Signals of both groups, call for presence of transcripts (Absent, A; Marginal, M; Present, P), p value for presence or absence of transcripts, log2 value of fold change (Signal Log Ratio, SLR) and the calculated fold change as mean values, call for the significance of differentially expression (Change Call: Increase, I; Marginal I, MI; Decrease, D; Marginal D, MD; No Change, NC), and the p value for that call. Additionally – not with GCOS calculated – t tests of log Signals and SLRs were included in the database. For each present transcript the significance of differential expression between the groups of arrays was either calculated using strict Bonferroni corrected Welch t tests between 9 SLR values of bone marrow vs. spleen and 12 SLR values within both groups (the latter 12 giving a mean SLR value of zero; p-value had to be <= 5.279E-08) or more than 50% of non parametrically calculated Change calls (Mann-Whitney U test, GCOS) i.e. 5 or more of all 9 Change calls have to be in the same direction. Significantly differentially expressed genes were filtered using the both default parameter sets of filter criteria; these are a combination of four different queries. Filter criteria were developed with various data sets of GeneChips and validated with the Affymetrix Latin Square dataset as shown in Menssen et al., 2009; PMID: 19265543.
Sample_platform_id	GPL1261
Sample_contact_name	Joachim,R.,Grün
Sample_contact_email	Gruen@DRFZ.de
Sample_contact_department	Bioinformatics
Sample_contact_institute	Deutsches Rheuma-Forschungszentrum (DRFZ)
Sample_contact_address	Charitéplatz 1
Sample_contact_city	Berlin
Sample_contact_zip/postal_code	D-10117
Sample_contact_country	Germany
Sample_contact_web_link	www.drfz.de
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM393nnn/GSM393941/suppl/GSM393941.CEL.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM393nnn/GSM393941/suppl/GSM393941.CHP.gz
Sample_series_id	GSE15733
Sample_data_row_count	45101

GSM393942

GPL1261

CD44highCD62L-CD25- CD4+ T cells from C57BL/6 murine spleen_rep1

CD44highCD62L-CD25- CD4+ T cells from C57BL/6 murine spleen

cell type: CD44highCD62L-CD25- CD4+ T cells tissue: spleen

Sample_geo_accession	GSM393942
Sample_status	Public on Apr 21 2009
Sample_submission_date	Apr 18 2009
Sample_last_update_date	Apr 20 2009
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_biomaterial_provider_ch1	C57BL/6 mice from DRFZ
Sample_treatment_protocol_ch1	CD44highCD62L-CD25- CD4+ T cells from spleen or bone marrow of 12-16 week old C57BL/6 mice were sorted with FACSAria
Sample_growth_protocol_ch1	n/a
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was extracted using Trizol reagent (Invitrogen). The amount, purity, and integrity of RNA was assessed for each sample using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip and a NanoDrop ND-1000 spectrophotometer. Contaminating genomic DNA was removed by an on-column DNA digestion step (Qiagen). Ten micrograms of total RNA was reverse-transcribed, followed by cDNA extraction using a PhaseLock gel (Eppendorf), and precipitation with ethanol and ammonium acetate.
Sample_label_ch1	biotin
Sample_label_protocol_ch1	10 µg of total RNA from each cell sample was reverse transcribed using T7-(d)T24 primer and SuperScript II reverse transcriptase, following cDNA extraction with a PhaseLock gel (Eppendorf), and precipitation with ethanol and ammonium acetate. Blocking was according to manufactures recommendations and probes were subsequently hybridized. After washing the hybridization signals were visualized by staining with streptavidin-phycoerythrin and amplification with anti-streptavidin.
Sample_hyb_protocol	Biotinylated cRNA was in vitro transcribed using the MEGAscript high yield transcription kit (Ambion) according to the manufacturer's recommendations. Biotinylated cRNA was fragmented, and the hybridization cocktail was prepared according to Affymetrix protocols (15 µg fragmented biotin-labeled cRNA spiked with Eukaryotic hybridization controls). The Murine Genome 430A version 2 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400.
Sample_scan_protocol	Arrays were scanned on an Affymetrix GeneChip Scanner 3000
Sample_data_processing	Data were analyzed according to High Performance Chip Data Analysis (HPCDA, unpublished - Joachim R. Gruen) with the Bioretis database (http://www.bioretis-analysis.de/) using the default filter parameters for decreased and increased gene lists (description of database c.f. open access paper Menssen et al., 2009; PMID: 19265543). Chip data included in the Bioretis database were analyzed using the GeneChip Operating Software (GCOS, Affymetrix), version 1.4. Microarrays were globally normalized and scaled to a trimmed mean expression value of 150. Quality checks were performed according to the manufacturer's recommendations. All 3 + 3 chips were compared to each other (bone marrow group vs. spleen, 9 comparisons, and all chips within the groups, twice 6 comparisons), and the following parameters of absolute and comparative analysis were included in the Bioretis database: expression heights (Signals) and mean, median and standard deviation of Signals of both groups, call for presence of transcripts (Absent, A; Marginal, M; Present, P), p value for presence or absence of transcripts, log2 value of fold change (Signal Log Ratio, SLR) and the calculated fold change as mean values, call for the significance of differentially expression (Change Call: Increase, I; Marginal I, MI; Decrease, D; Marginal D, MD; No Change, NC), and the p value for that call. Additionally – not with GCOS calculated – t tests of log Signals and SLRs were included in the database. For each present transcript the significance of differential expression between the groups of arrays was either calculated using strict Bonferroni corrected Welch t tests between 9 SLR values of bone marrow vs. spleen and 12 SLR values within both groups (the latter 12 giving a mean SLR value of zero; p-value had to be <= 5.279E-08) or more than 50% of non parametrically calculated Change calls (Mann-Whitney U test, GCOS) i.e. 5 or more of all 9 Change calls have to be in the same direction. Significantly differentially expressed genes were filtered using the both default parameter sets of filter criteria; these are a combination of four different queries. Filter criteria were developed with various data sets of GeneChips and validated with the Affymetrix Latin Square dataset as shown in Menssen et al., 2009; PMID: 19265543.
Sample_platform_id	GPL1261
Sample_contact_name	Joachim,R.,Grün
Sample_contact_email	Gruen@DRFZ.de
Sample_contact_department	Bioinformatics
Sample_contact_institute	Deutsches Rheuma-Forschungszentrum (DRFZ)
Sample_contact_address	Charitéplatz 1
Sample_contact_city	Berlin
Sample_contact_zip/postal_code	D-10117
Sample_contact_country	Germany
Sample_contact_web_link	www.drfz.de
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM393nnn/GSM393942/suppl/GSM393942.CEL.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM393nnn/GSM393942/suppl/GSM393942.CHP.gz
Sample_series_id	GSE15733
Sample_data_row_count	45101

GSM393943

GPL1261

CD44highCD62L-CD25- CD4+ T cells from C57BL/6 murine spleen_rep2

CD44highCD62L-CD25- CD4+ T cells from C57BL/6 murine spleen

cell type: CD44highCD62L-CD25- CD4+ T cells tissue: spleen

Sample_geo_accession	GSM393943
Sample_status	Public on Apr 21 2009
Sample_submission_date	Apr 18 2009
Sample_last_update_date	Apr 20 2009
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_biomaterial_provider_ch1	C57BL/6 mice from DRFZ
Sample_treatment_protocol_ch1	CD44highCD62L-CD25- CD4+ T cells from spleen or bone marrow of 12-16 week old C57BL/6 mice were sorted with FACSAria
Sample_growth_protocol_ch1	n/a
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was extracted using Trizol reagent (Invitrogen). The amount, purity, and integrity of RNA was assessed for each sample using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip and a NanoDrop ND-1000 spectrophotometer. Contaminating genomic DNA was removed by an on-column DNA digestion step (Qiagen). Ten micrograms of total RNA was reverse-transcribed, followed by cDNA extraction using a PhaseLock gel (Eppendorf), and precipitation with ethanol and ammonium acetate.
Sample_label_ch1	biotin
Sample_label_protocol_ch1	10 µg of total RNA from each cell sample was reverse transcribed using T7-(d)T24 primer and SuperScript II reverse transcriptase, following cDNA extraction with a PhaseLock gel (Eppendorf), and precipitation with ethanol and ammonium acetate. Blocking was according to manufactures recommendations and probes were subsequently hybridized. After washing the hybridization signals were visualized by staining with streptavidin-phycoerythrin and amplification with anti-streptavidin.
Sample_hyb_protocol	Biotinylated cRNA was in vitro transcribed using the MEGAscript high yield transcription kit (Ambion) according to the manufacturer's recommendations. Biotinylated cRNA was fragmented, and the hybridization cocktail was prepared according to Affymetrix protocols (15 µg fragmented biotin-labeled cRNA spiked with Eukaryotic hybridization controls). The Murine Genome 430A version 2 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400.
Sample_scan_protocol	Arrays were scanned on an Affymetrix GeneChip Scanner 3000
Sample_data_processing	Data were analyzed according to High Performance Chip Data Analysis (HPCDA, unpublished - Joachim R. Gruen) with the Bioretis database (http://www.bioretis-analysis.de/) using the default filter parameters for decreased and increased gene lists (description of database c.f. open access paper Menssen et al., 2009; PMID: 19265543). Chip data included in the Bioretis database were analyzed using the GeneChip Operating Software (GCOS, Affymetrix), version 1.4. Microarrays were globally normalized and scaled to a trimmed mean expression value of 150. Quality checks were performed according to the manufacturer's recommendations. All 3 + 3 chips were compared to each other (bone marrow group vs. spleen, 9 comparisons, and all chips within the groups, twice 6 comparisons), and the following parameters of absolute and comparative analysis were included in the Bioretis database: expression heights (Signals) and mean, median and standard deviation of Signals of both groups, call for presence of transcripts (Absent, A; Marginal, M; Present, P), p value for presence or absence of transcripts, log2 value of fold change (Signal Log Ratio, SLR) and the calculated fold change as mean values, call for the significance of differentially expression (Change Call: Increase, I; Marginal I, MI; Decrease, D; Marginal D, MD; No Change, NC), and the p value for that call. Additionally – not with GCOS calculated – t tests of log Signals and SLRs were included in the database. For each present transcript the significance of differential expression between the groups of arrays was either calculated using strict Bonferroni corrected Welch t tests between 9 SLR values of bone marrow vs. spleen and 12 SLR values within both groups (the latter 12 giving a mean SLR value of zero; p-value had to be <= 5.279E-08) or more than 50% of non parametrically calculated Change calls (Mann-Whitney U test, GCOS) i.e. 5 or more of all 9 Change calls have to be in the same direction. Significantly differentially expressed genes were filtered using the both default parameter sets of filter criteria; these are a combination of four different queries. Filter criteria were developed with various data sets of GeneChips and validated with the Affymetrix Latin Square dataset as shown in Menssen et al., 2009; PMID: 19265543.
Sample_platform_id	GPL1261
Sample_contact_name	Joachim,R.,Grün
Sample_contact_email	Gruen@DRFZ.de
Sample_contact_department	Bioinformatics
Sample_contact_institute	Deutsches Rheuma-Forschungszentrum (DRFZ)
Sample_contact_address	Charitéplatz 1
Sample_contact_city	Berlin
Sample_contact_zip/postal_code	D-10117
Sample_contact_country	Germany
Sample_contact_web_link	www.drfz.de
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM393nnn/GSM393943/suppl/GSM393943.CEL.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM393nnn/GSM393943/suppl/GSM393943.CHP.gz
Sample_series_id	GSE15733
Sample_data_row_count	45101

GSM393944

GPL1261

CD44highCD62L-CD25- CD4+ T cells from C57BL/6 murine spleen_rep3

CD44highCD62L-CD25- CD4+ T cells from C57BL/6 murine spleen

cell type: CD44highCD62L-CD25- CD4+ T cells tissue: spleen

Sample_geo_accession	GSM393944
Sample_status	Public on Apr 21 2009
Sample_submission_date	Apr 18 2009
Sample_last_update_date	Apr 20 2009
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_biomaterial_provider_ch1	C57BL/6 mice from DRFZ
Sample_treatment_protocol_ch1	CD44highCD62L-CD25- CD4+ T cells from spleen or bone marrow of 12-16 week old C57BL/6 mice were sorted with FACSAria
Sample_growth_protocol_ch1	n/a
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was extracted using Trizol reagent (Invitrogen). The amount, purity, and integrity of RNA was assessed for each sample using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip and a NanoDrop ND-1000 spectrophotometer. Contaminating genomic DNA was removed by an on-column DNA digestion step (Qiagen). Ten micrograms of total RNA was reverse-transcribed, followed by cDNA extraction using a PhaseLock gel (Eppendorf), and precipitation with ethanol and ammonium acetate.
Sample_label_ch1	biotin
Sample_label_protocol_ch1	10 µg of total RNA from each cell sample was reverse transcribed using T7-(d)T24 primer and SuperScript II reverse transcriptase, following cDNA extraction with a PhaseLock gel (Eppendorf), and precipitation with ethanol and ammonium acetate. Blocking was according to manufactures recommendations and probes were subsequently hybridized. After washing the hybridization signals were visualized by staining with streptavidin-phycoerythrin and amplification with anti-streptavidin.
Sample_hyb_protocol	Biotinylated cRNA was in vitro transcribed using the MEGAscript high yield transcription kit (Ambion) according to the manufacturer's recommendations. Biotinylated cRNA was fragmented, and the hybridization cocktail was prepared according to Affymetrix protocols (15 µg fragmented biotin-labeled cRNA spiked with Eukaryotic hybridization controls). The Murine Genome 430A version 2 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400.
Sample_scan_protocol	Arrays were scanned on an Affymetrix GeneChip Scanner 3000
Sample_data_processing	Data were analyzed according to High Performance Chip Data Analysis (HPCDA, unpublished - Joachim R. Gruen) with the Bioretis database (http://www.bioretis-analysis.de/) using the default filter parameters for decreased and increased gene lists (description of database c.f. open access paper Menssen et al., 2009; PMID: 19265543). Chip data included in the Bioretis database were analyzed using the GeneChip Operating Software (GCOS, Affymetrix), version 1.4. Microarrays were globally normalized and scaled to a trimmed mean expression value of 150. Quality checks were performed according to the manufacturer's recommendations. All 3 + 3 chips were compared to each other (bone marrow group vs. spleen, 9 comparisons, and all chips within the groups, twice 6 comparisons), and the following parameters of absolute and comparative analysis were included in the Bioretis database: expression heights (Signals) and mean, median and standard deviation of Signals of both groups, call for presence of transcripts (Absent, A; Marginal, M; Present, P), p value for presence or absence of transcripts, log2 value of fold change (Signal Log Ratio, SLR) and the calculated fold change as mean values, call for the significance of differentially expression (Change Call: Increase, I; Marginal I, MI; Decrease, D; Marginal D, MD; No Change, NC), and the p value for that call. Additionally – not with GCOS calculated – t tests of log Signals and SLRs were included in the database. For each present transcript the significance of differential expression between the groups of arrays was either calculated using strict Bonferroni corrected Welch t tests between 9 SLR values of bone marrow vs. spleen and 12 SLR values within both groups (the latter 12 giving a mean SLR value of zero; p-value had to be <= 5.279E-08) or more than 50% of non parametrically calculated Change calls (Mann-Whitney U test, GCOS) i.e. 5 or more of all 9 Change calls have to be in the same direction. Significantly differentially expressed genes were filtered using the both default parameter sets of filter criteria; these are a combination of four different queries. Filter criteria were developed with various data sets of GeneChips and validated with the Affymetrix Latin Square dataset as shown in Menssen et al., 2009; PMID: 19265543.
Sample_platform_id	GPL1261
Sample_contact_name	Joachim,R.,Grün
Sample_contact_email	Gruen@DRFZ.de
Sample_contact_department	Bioinformatics
Sample_contact_institute	Deutsches Rheuma-Forschungszentrum (DRFZ)
Sample_contact_address	Charitéplatz 1
Sample_contact_city	Berlin
Sample_contact_zip/postal_code	D-10117
Sample_contact_country	Germany
Sample_contact_web_link	www.drfz.de
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM393nnn/GSM393944/suppl/GSM393944.CEL.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM393nnn/GSM393944/suppl/GSM393944.CHP.gz
Sample_series_id	GSE15733
Sample_data_row_count	45101

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