GSM8709996: 14_235074V03-0_EM; Homo sapiens; RNA-Seq
Cells were sorted on a FACS Aria III (BD, Franklin Lakes, NJ). Markers used for sorting are shown in supplementary table 3. Cells were sorted into CD4+ Tnaive (CCR7+CD45RA+), TEM (CCR7-CD45RA-), and TCM (CCR7+CD45RA-) subsets. Total CD4+ T cells refers to data combining normalized gene expression from these subsets. An average of 15,000 cells were sorted at 4° C into RNAse-free eppendorf tubes containing RPMI. Cells were then centrifuged at 600 x g for 10 minutes and supernatant was removed. 350uL RLT buffer (Qiagen, Hilden, Germany) plus 1% BME was added, and cells were vortexed for 1 min. Cells were lysed in 350 μL of Buffer RLT and then extracted using the RNeasy Micro kit (Qiagen) with on-column DNase digestion. RNA quality was assessed using a Bioanalyzer 2100 (Agilent) and then 500 picograms of total RNA was used as input for cDNA synthesis using the Clontech SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio) according to the manufacturer's instructions. Amplified cDNA was fragmented and appended with dual-indexed barcodes using the Nextera XT DNA Library Preparation kit (Illumina). Libraries were validated by capillary electrophoresis on a TapeStation 4200 (Agilent), pooled at equimolar concentrations, and sequenced with SE100 reads on an Illumina NovaSeq 6000, yielding ~25 million reads per sample on average.