GSM5385387: 496_1_0336_PBMCs; Homo sapiens; RNA-Seq
PBMCs were stained with anti-human CD3, CD4, CD8, CD14, CD19, CD56, and live/dead viability antibodies (see Supplemental Table S2 for antibody details). Subsequently, live CD4+ single positive (SP) (CD14-CD19-CD56-CD3+CD4+CD8-), CD8+ SP (CD14-CD19-CD56-CD3+CD4-CD8+), and CD4+CD8+ DP (CD14-CD19-CD56-CD3+CD4+CD8+) cells were sorted into TRIzol LS Reagent (Invitrogen, Carlsbad, CA) using a BD FACSAria cell sorter (BD Biosciences, San Jose, CA). 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 (5 ng) was amplified following the Smart-seq2 protocol. cDNA was purified using AMPure XP beads (1:1 ratio; Beckman Coulter, Carlsbad, CA). From this step, 1 ng cDNA was used to prepare a standard Nextera XT sequencing library (Nextera XT DNA library preparation kit and index kit (set B and C, respectively), Illumina, San Diego, CA).