GSM3896853: MR1tet- T cells, donor TU0063; Homo sapiens; RNA-Seq
For each condition, 200 cells were collected at 4C in 8 µl of lysis buffer (0.2% Triton X-100, 2U/µl of recombinant RNase inhibitor (Clontech/Takara), 5 mM dNTP mix (Life Technologies) in a 96-well PCR plate (Bio-Rad). Immediately after sorting, plates were spun for 1 min at 3000 rpm, and stored at -80C until RNA extraction. Four microliters of each sample was amplified following the Smart-seq2 protocol. mRNA was captured using poly-dT oligonucleotides and directly reverse transcribed into full-length cDNA (amplified by PCR for 16 cycles). cDNA was purified using AMPure XP beads (Beckman Coulter). From this step, 1 ng of cDNA was used to prepare a standard Nextera XT sequencing library (Nextera XT DNA sample preparation kit and index kit, Illumina). Whole-transcriptome amplification and sequencing library preparations were performed in a 96-well format to reduce assay-to-assay variability. Barcoded Illumina sequencing libraries (Nextera, Illumina) were generated using the automated platform (Biomek FXp). Libraries were sequenced on a HiSeq 2500 Illumina platform to obtain 50-bp single-end reads (TruSeq Rapid kit; Illumina).