GSM4859874: PB CD8+CD45RO+ T cell 7; Homo sapiens; RNA-Seq
Mononuclear cells from the peripheral blood (PBMCs) were isolated by Ficoll density‐gradient centrifugation and subsequent collection of the leukocyte-containing interphase. For the isolation of leukocytes from the synovial fluid, we diluted the viscous fluid with 5 mM ethylenediaminetetraacetic acid (EDTA)-containing phosphate-buffered saline (PBS) and filtered the cells using a 70 μm cell strainer (BD). Subsequently, cells were centrifuged at 300 g at 4 °C and resuspended in PBS containing 0.2% bovine serum albumin (PBS/BSA). Following an additional filtration step through a 70 μm cell strainer, cells were resuspended in PBS/BSA prior to depletion of CD15+ neutrophils by magnetic cell separation (MACS, Miltenyi Biotec). Single cell suspensions of PBMCs and CD15-depleted SF cells were blocked with human FcR blocking reagent (Miltenyi Biotec) and labeled for 15 min at 4 °C with antibodies for fluorescent activated cell sorting (FACS) using a FACS-Aria II (BD). Cells were sorted to obtain antigen-experienced (memory) CD4+CD45RO+CD25- T cells (Tcon), CD4+CD45RO+CD127loCD25+ regulatory T cells (Treg), and CD8+CD45RO+ T cells. Sorted cells then were subjected to single-cell sequencing by applying them to the 10X Genomics platform. For single cell library preparation, FACS-sorted human T cells were applied to the 10X Genomics platform using the Chromium Single Cell 5' Library & Gel Bead Kit (10x Genomics) and following the manufacturer's instructions for capturing ~3000 cells. The amplified cDNA was used for simultaneous 5' gene expression (GEX) and TCR library preparation. TCR target enrichment was achieved using the Chromium Single Cell V(D)J Enrichment Kit for Human T cells. Through fragmentation, adapter ligation and index PCR the final libraries were obtained. The quality of single cell 5' GEX and TCR libraries was assessed by Qubit quantification, Bioanalyzer fragment analysis (HS DNA Kit, Agilent) and KAPA library quantification qPCR (Roche). The sequencing was performed on a NextSeq500 device (Illumina) using High Output v2 Kits (150 cycles) with the recommended sequencing conditions for 5' GEX libraries (read1: 26nt, read2: 98nt, index1: 8nt, index2: n.a.) and Mid Output v2 Kits (300 cycles) for TCR libraries (read1: 150nt, read2: 150nt, index1: 8nt, index2: n.a., 20% PhiX spike-in). Raw Illumina-NextSeq 500 data were processed using cellranger-2.1.1. Mkfastq, count commands and vdj commands were used with default parameter settings. The genome reference was refdata-cellranger-hg19-1.2.0. The vdj-reference was refdata-cellranger-vdj-GRCh38-alts-ensembl-2.0.0. In both cases, the number of expected cells was set to 3000.