GSM3896816: MR1tet+ CD8+ T cells, donor TP0018; Homo sapiens; RNA-Seq
Total RNA was purified using an miRNeasy Micro Kit (QIAGEN) and quantified by quantitative PCR. Purified total RNA (1-5 ng) 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).