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Status |
Public on May 18, 2015 |
Title |
E6 |
Sample type |
SRA |
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Source name |
CCR6+, CD4+ T cells from HLA-DR4+ healthy control, myelin tetramer positive
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Organism |
Homo sapiens |
Characteristics |
diagnosis: Healthy cell population: CCR6+, CD4+ T cells from HLA-DR4+ healthy control, myelin tetramer positive
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Treatment protocol |
Amplified T cell libraries assay were carried out as previous described (Geiger, 2009). Naïve, CCR6− and CCR6+ memory CD4+ T from paired MS patients and healthy controls were pre-sorted and cultured in 96-well round-bottom plates (Costar) at 2 × 103 cells per well in complete RPMI 1640 medium, and stimulated with 1 μg/ml PHA (Roche) and 20 U/ml IL-2 in the presence of irradiated (45Gy) allogeneic feeder cells (2×104/well). IL-2 was added on day 4, 7 and 10. Cultures were washed and split into two 96-well plates after 2-weeks of stimulation and expansion. Library screening was performed by culturing ~ 106 T cells/well with autologous monocytes (~105), which were either unpulsed or pulsed for 3 h with 10 μg/ml myelin peptide pools (MBP85-99, MOG222-241, PLP30-49, PLP129-148, MOG97-109, PLP180-199), or C. albicans (GREER). [3H] (Perkin Elmer) was added into the cultures 16 h before harvest. On day 5, cell proliferation was measured by 3H-thymidine incorporation on a scintillation beta-counter (Perkin Elmer). Culture supernatants were taken on day 7 for cytokine profiling as described below.
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Growth protocol |
CCR6+ memory CD4+ T cells from HLA-DR4+ healthy controls and HLA-DR4+ MS patients were amplified by PHA and IL-2 and stimulated by irradiated autologous monocytes and DR4 myelin peptides (MOG97-109 and PLP180-199). Cell proliferation was measured and two of the highest proliferated wells were picked for DR4 tetramers staining (MOG97-109-tetramers and PLP180-199-tetramers). Myelin tetramer+ and tetramer− cells were sorted into RNA lysis buffer for RNA sequencing.
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Extracted molecule |
total RNA |
Extraction protocol |
RNA was extracted using the NucleoSpin RNA XS Kit (Macherey-Nagel, Bethlehem, PA) according to the manufacturers instructions. cDNA synthesis and amplification were performed using SMARTer Ultra Low Input RNA for Illumina Sequencing High Volume Kit (Clontech, Mountain View, CA) according to the manufacturers instructions. The average number of cells used to isolate RNA was 3,000, which yielded >1ng input RNA. Paired-end sequencing libraries were prepared using the Nextera XT DNA sample Prep Kit (Illumina, SanDiego, CA) according to the manufacturers instructions.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
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Description |
HC3_Tet+1 RNA-Seq followed by Nextera XT
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Data processing |
Hiseq 2500 processing Alignments done with Tophat v1.4.1 to hg19 Cufflinks v2.1.1 used to summarize expression with the --compatible-hits-norm option and the annotation file -G Homo_sapiens.GRCh37.74.gtf Genome_build: hg19 Supplementary_files_format_and_content: gtf annotation file for cufflinks transcripts Supplementary_files_format_and_content: tab-delimited text file with transcript ids described by bathomas_merged.gtf, gene symbol and 16 sample columns containing log2 RPKM values for each sample
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Submission date |
Mar 10, 2015 |
Last update date |
May 15, 2019 |
Contact name |
Charles Arthur Whittaker |
E-mail(s) |
charliew@mit.edu
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Organization name |
Koch Institute
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Street address |
77 Mass Ave 76-189
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City |
Cambridge |
State/province |
MA |
ZIP/Postal code |
02152 |
Country |
USA |
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Platform ID |
GPL16791 |
Series (1) |
GSE66763 |
Functional Inflammatory Profiles Distinguish Myelin-Reactive T Cells from Patients with Multiple Sclerosis |
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Relations |
BioSample |
SAMN03397782 |
SRA |
SRX950766 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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