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Sample GSM1577739

Query DataSets for GSM1577739

Status

Public on Feb 17, 2015

Title

Effector 3h

Sample type

SRA

Source name

T cells

Organism

Homo sapiens

Characteristics

cell state: Effector 3h

Treatment protocol

Cells were stimulated in 6-well plates at 8x106 cells/2 ml with soluble aCD3 (2 mg/ml, OKT-3, BioXCell) and aCD28 (1 mg/ml, BD Biosciences) or CTLA4-Ig (7.5 mg/ml, BioXCell).

Growth protocol

Blood samples were obtained from Hoxworth Blood bank. Samples were de-identified and the study was done under an exemption provided by CCHMC IRB. PBMCs were depleted of CD45RO+, CD8+, and CD25+ cells using biotinylated antibodies (BioLegend) and IMagTM streptavidin beads (BD Biosciences) and stimulated in 6-well plates at 8x106 cells/2 ml with soluble aCD3 (2 mg/ml, OKT-3, BioXCell) and aCD28 (Effector samples; 1 mg/ml, BD Biosciences) or CTLA4-Ig (Anergic samples; 7.5 mg/ml, BioXCell) in RPMI 1640 supplemented with 10% FBS, penicillin/streptomycin, L-glutamine, and b-mercapethanol. At specified timepoints CD4 T cells were purified using Invitrogen positive selection kit.

Extracted molecule

polyA RNA

Extraction protocol

RNA was extracted with Aurum™ Total RNA Mini Kit (Bio-Rad)
A modified dUTP method was used for preparation of RNA-Seq libraries[27]. Briefly, mRNA was purified from total RNA by Dynabeads® mRNA Purification Kit (Invitrogen) and reverse-transcribed with SuperScript® III (Invitrogen). The second strand was created by DNA Polymerase 1 (NEB) in the presence of RNase H and a dNTP mix containing dUTP instead dTTP. The cDNA was fragmented on a Covaris sonicator, and end repair and A-tailing were performed using the Quick Blunting kit and exo- Klenow fragment of DNA polymerase I, respectively (NEB). Illumina genomic adapters were ligated, and the sample was size-fractionated (~170–400 bp) on E-Gel EX 2% agarose (Invitrogen). After uracil-dna glycosylase (UDG) treatment (NEB) and a final PCR amplification step with Illumina primers (18 cycles), the cDNA libraries were size-fractionated (~175-400 bp) on a 2% agarose gel, quantified using a Qubit 2.0 fluorometer and dsDNA HS kit (Invitrogen), and sent to sequencing on a Illumina HiSeq 2500 sequencing system

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

RNA-Seq data analysis was performed using the Wardrobe Experiment Management System (http://www.biorxiv.org/content/early/2014/12/18/012799; https://code.google.com/p/genome-tools/).
Briefly, reads were mapped to the hg19 genome and RefSeq-based transcriptome using RNA-STAR (v. 2.4.0c and assigned to the RefSeq genes using the Wardrobe algorithm.
STAR PARAMS:
--clip3pNbases 0 --seedSearchStartLmax 15 --alignSJDBoverhangMin 1
--outFilterMultimapNmax 1 --outFilterMismatchNmax 5 --clip5pNbases 0
Genome_build: hg19
Supplementary_files_format_and_content: csv file includes RPKM values for each sample

Submission date

Jan 06, 2015

Last update date

May 15, 2019

Contact name

Artem Barski

Organization name

Cincinnati Children's Hospital

Department

Allergy

Street address

3333 Burnet Av