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Sample GSM1631666

Query DataSets for GSM1631666

Status

Public on May 18, 2015

Title

Sample type

SRA

Source name

CCR6+, CD4+ T cells from HLA-DR4+ healthy control, myelin tetramer negative

Organism

Homo sapiens

Characteristics

diagnosis: Healthy
cell population: CCR6+, CD4+ T cells from HLA-DR4+ healthy control, myelin tetramer negative

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.

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.

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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

HC3_Tet-1
RNA-Seq followed by Nextera XT

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

Submission date

Mar 10, 2015

Last update date

May 15, 2019

Contact name

Charles Arthur Whittaker

E-mail(s)

charliew@mit.edu

Organization name

Koch Institute

Street address

77 Mass Ave 76-189