GSM2332818: GS0053_CD4_Sri Lanka_negative; Homo sapiens; RNA-Seq
Cryopreserved PBMC were thawed, stained with antibodies, and 100,000 memory CD4 or memory CD8 T cells sorted using BD Aria 3 or BD Aria Fusion cell sorter direclty in Trizol. Memory cells were defined as the combination of CCR7-CD45RA-, CCR7-CD45RA+ and CCR7+CD45RA- populations. Total RNA was purified using a miRNAeasy micro kit (Qiagen, USA) and quantified by qPCR as described previously (Seumois et al., 2012). Purified total RNA (1 to 5 ng) was amplified following the smart-seq2 protocol (16 cycles of cDNA amplification) (Picelli et al., 2014). 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).