GSM9284687: sorted Tscm cells, HC3; Homo sapiens; RNA-Seq
Peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation. Cells were washed with PBS, stained with Tscm marker dye (CD3, CCR7, CD45RA, CD27, CD95), washed and resuspended in FACS buffer (1% v/v FBS in PBS). Tscm cells were sorted (CD3+CCR7+CD45RA+CD27+CD95high) by fluorescence-activated cell sorting (FACS) using a FACSAria III instrument (BD Biosciences). The sorted Tscm cells were washed in FACS buffer and subsequently used for single-cell RNA sequencing. Cells were loaded into a Chromium Single Cell Controller (10x Genomics) using either the Chromium Single Cell 5′ Reagent Kit v1.1 or v2.0, targeting for a cell recovery of 10,000 cells per sample. Gel bead-in-emulsion (GEM) generation, barcoding, reverse transcription, and cDNA amplification were performed according to the manufacturer's protocol to construct 5′ gene expression libraries. Libraries were sequenced on an Illumina NovaSeq 6000 platform. Raw base call files were converted to FASTQ format using bcl2fastq2.