GSM7519041: P1_HC_sc_library; Homo sapiens; RNA-Seq
Human peripheral blood from the patient (P1) or two age-matched and sex-matched controls were collected in sodium heparin tubes and peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation. CD3+ T cells were enriched from PBMC using an EasySep Human T cell isolation kit (#1791, Stemcell Technologies) and sorted for CD4+CD25+CD127lo regulatory T cells (Tregs) or CD4+CD25-CD127+ conventional T cells (Tconvs). Tregs and Tconvs were either left untreated or stimulated with PMA+ionomycin for 4 hours before RNA extraction. The samples underwent reverse transcription, Exonuclease I treatment, and cDNA amplification. mRNA PCR products were separated from sample tag products with double-sided size selection using AMPure beads. Separated products were further amplified using PCR and then purified. PCR products were then PCR amplified using the Illumina i7 index primer. Libraries were diluted to 650pM and multiplexed for paired-end (2 x 115bp) sequencing on a NextSeq2000 Sequencing System (Illumina) at 200 cycles P3 for 1.1 billion reads.