GSM7015711: Donor 3 (S3), CD4+ isolated post colture (POSTc), RPMI, unstimulated (US); Homo sapiens; RNA-Seq
CD4+ T cells were first lysed in a solution of RNeasy Lysis Buffer (RLT Buffer, QIAGEN) and beta-mercaptoethanol (BME, Gibco REF #21985-023, using 10 ul BME for each ml of RLT) and then stored at -20° overnight for RNA yields maximization. The day after, according to cell number availability, RNA isolation was made via the RNeasy™ Mini and Micro Kit™ (QIAGEN, Cat. #80204 and #80284 respectively) following manufacturers' instructions. Pure, concentrated RNA was eluted in 14 ul (Micro kit) or 30 ul (Mini kit) RNase-free water and 1ul of RNAse Inhibitor (Cat. #AM2694, InvitrogenTM) was added to avoid sample degradation. The PCR-cDNA Sequencing Kit SQK-PCS109 was used for S1 PREc samples. Meanwhile, the PCR-cDNA Barcoding kit SQK-PCB109 was used to multiplex the samples of each donor in a single library. The sequencing libraries were prepared following the manufacturer's protocol. In brief, starting from 50 ng of full-length RNA, complementary strand DNA (cDNA) synthesis and strand switching were performed using kit-supplied oligonucleotides. dscDNA was generated by PCR amplification of cDNA (10ul cDNA/sample) using primers with 5' tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters. The prepared libraries were either immediately used for loading onto the R9.4.1 flow cell (FLO-MIN106) and sequenced or stored at -80°C until use. Libraries were run on MinION flow cells with MinKNOW acquisition software version v.21.02.01.