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Sample GSM2464962

Query DataSets for GSM2464962

Status

Public on Mar 18, 2019

Title

Sample_deg_0hrs

Sample type

SRA

Source name

Murine Adult Spleen CD4 T Cells

Organism

Mus musculus

Characteristics

strain: C57BL/6
developmental stage: Murine Adult CD4 T cells cultured in vitro with Actinomysin
culture condition: Stimulated for 16 hours and than Actinomysin added
genotype: Wildtype

Treatment protocol

CD4 Th cells were purified and set up in culture under three different Th differentiation conditions: Th0 condition was set up by treating the purified CD4 cells with anti-cd3e and anti-cd28; Th1 condition was set up with anti-cd3e, anti-cd28, anti-IL-4 and recombinant IL-12; and Th2 condition was set up with anti-cd3e, anti-cd28, anti-IL-12, anti-IFNG and recombinant IL-4.

Extracted molecule

total RNA

Extraction protocol

Murine CD4 positive cells were isolated from spleen cells by magnetic bead separation using EasySep mouse CD4+ cell negative selection kit (StemCell Technologies) according to the manufacturer’s instructions. The purity of isolated CD4+ cells was routinely exceeded 95%. For activation, CD4+cells were cultured at 5x106 cells/ml coated with 5µg/ml anti-CD3, and added 1µg/ml soluble anti-CD28 in complete RPMI and analysed at 4 and 20h. Th0, Th1 and Th2 skewing cultures, CD4+ cells were cultured on 5ug/ml plate bound anti-CD3 with were culture with 1µg/ml soluble anti-CD28 in complete RPMI. Th1 conditions the solution containing 5µg/ml anti-IL-4 and 10ng/ml rmIL-12. For Th2 cultures, 5µg/ml anti-IFNɣ; 20ng/ml rmIL-4, and 5µg/ml anti-IL-12 were added and, Cells were cultured at 37 °C under 5% CO2. Cells were harvested 72h and T-bet and Gata-3 were analysed through intracellular staining assay as described (Furmanski et al. 2013) (antibodies/proteins: eBioscience).
RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

Deg 0hours

Data processing

Base Calling was done using the RTA package from Illumina
Alignment was done on the transcriptome using the Burrows-Wheeler Aligner (BWA) package
Expression levels in Reads Per Million Kilobases (RPKM).
Genome_build: GRCm38
Supplementary_files_format_and_content: comma separated file including RPKM values for each Sample.

Submission date

Jan 22, 2017

Last update date

May 15, 2019

Contact name

Tessa Crompton

Organization name

UCL

Department

Immunobiology

Street address

30 Guilford Street