GSM1357216: RNA-Seq-Th1-Donor2; Homo sapiens; RNA-Seq
From PBMC fraction of leukapheresis pouch, CD4 T cells were purified using the Miltenyi T cell isolation kit II according to manufacturer's instructions. Purified cells were incubated in PBS containing 0.5% BSA and 2 mM EDTA pH 8.0 (MACS buffer) with a dilution of class II tetramer (10ml tetramer per 50x106 CD4 T cells) for 2 h at room temperature (HLA class II tetramers conjugated using PE labeled streptavidin were provided by the Tetramer Core Laboratory at Benaroya Research Institute).  Cells were then stained for cell surface antigens using anti-CD4-APC EFluor780 (RPA-T4), anti-CD45RA-EFluor450 (HI100) (both from Affymetrix eBioscience), anti-CD3-Alexa Fluor 700 (UCHT1), anti-CXCR3(CD183)-APC or CXCR3-Alexa Fluor 488 (1C6/CXCR3), anti-CD19-V500 (HIB19), anti-CD14-V500 (M5E2), anti-CD8-V500 (RPA-T8), anti-CD25-FITC (M-A251), anti-CCR4-PECy7 or CCR4-PE (1G1), anti-CCR6(CD196)-biotinylated (11A9) (all from BD Biosciences) followed by streptavidin-BV605 (BioLegend), anti-CCR7(CD197)-PerCPCy5.5 (G043H7) (BioLegend) and Live/Dead Aqua (Affymetrix eBiosciences) to exclude dead cells. Cells were sorted into six separate CD4+CD3+CD8/14/19−CD25- populations; tetramer+, CXCR3+CCR6+CCR4− (Th*), CXCR3+CCR6−CCR4− (Th1), CXCR3−CCR6+CCR4− (Th17), CXCR3−CCR6−CCR4+ (Th2) and CD4+CD3+CD8/14/19− CD45RA+CD25−CCR7+CXCR3−CCR6−CCR4− (naïve) cells directly in Trizol LS reagents. For RNAseq of activated cells; Th* cells were sorted from purified CD4+ T cells on a BD Aria flow cytometer. These and remaining cells (used as antigen presenting cells, APC) were rested over night in complete RPMI medium at 37°C in 5% CO2. APCs were stained with CFSE according to manufacturer's instruction (Affymetrix eBioscience). Cells were cultured at a ratio of 2:1 CD4+ T cells:APC in the presence of 5 μg/ml MTB peptide pool or 0.1 μg/ml PMA with 1 μg/ml Ionomycin for 6 h in complete RPMI medium at 37°C in 5% CO2. Unstimulated cells were used to assess nonspecific/background activation. After 6 h, cells were harvested and CFSE negative cells (Th*) were sorted from the APCs on a BD Aria flow cytometer directly in Trizol LS cell lysis reagent. RNA was extracted using miRNease kit from Qiagen following manufacturer's instructions. Total RNA was purified using miRNAeasy kit (Qiagen) and quantified as described previously (Seumois et al, 2012). 10-15ng of purified total RNA was used for poly A section (Poly(A)Purist Mag kit, Lifetechnologies). Poly A selected RNA was amplified with the Whole Transcriptome Amplification Sequencing Technology kit (SEQR, Sigma) as per manufacturers recommendation. One μg of this amplified cDNA was treated with restriction enzyme (SEQR, Sigma), to remove the primer sequences and then purified using AmpureXP beads (Beckman Coulter). Efficiency of removal of SEQR primer sequences was assessed by PCR. From this step, 250 ng of purified DNA was diluted with Te to obtain a total volume of 65 ul. Diluted cDNA was sonicated with E220 Covaris multiplex sonicator (Covaris) to generate 100-250bp DNA fragments. ~ 250 ng of DNA was used for preparing standard SOLiD sequencing library (5500 SOLiD® Fragment 48 Library Core Kit & Fragment Library Barcode Adaptors 1-96). Following emulsion PCR samples were sequenced on the SOLiD 5500 sequencer to obtain 35bp single end reads (SOLiD™ EZ Bead™ E120 System kits). Both, whole transcriptome amplification and sequencing library preparation were performed in a 96-well format, which significantly reduced hands-on time, besides reducing technical and assay-to-assay variability.