GSM4498551: CB_donor1_γδT_transcriptome; Homo sapiens; RNA-Seq
Peripheral blood mononuclear cells (PBMCs) and cord blood mononuclear cells (CBMCs) were isolated from fresh blood samples stored in EDTA using a density gradient media (Pancoll, PAN-Biotech). After isolation, mononuclear cells were washed twice in PBS 10% FBS (Sigma-Aldrich) and cryopreserved at -80°C in freezing medium (50% FBS (Sigma-Aldrich), 40% RPMI 1640 (Gibco), 10% DMSO (Roth)). Cells were cultured overnight in X-Vivo 15 medium (Lonza) before antibody staining and cell sorting and treated with 5% FcR block in buffer (PBS with 3% FCS, 4 mM EDTA) right before staining. Cells were stained with hCD3 FITC (1:100; clone BW264/56; Miltenyi), hTCR γδ PE (1:50; clone 11F2; Miltenyi), and DAPI (0.1 μg/ml) to determine living cells. For sequencing experiments, DAPI−, CD3+, γδ+ T cells were sorted on a FACS Aria Fusion cytometer (BD) and subsequently used for the preparation of single-cell libraries using the single-cell immune profiling platform of 10x Genomics. scRNA-seq and single-cell TCR libraries were prepared using the Chromium Single Cell V(D)J Reagent Kit (10x Genomics). For each sample, 3*10e4 γδ T cells mixed with a master mix for cDNA generation were used in a droplet-based approach for the generation of Gel Bead-in-emulsion (GEMs). 5' Gel beads contain adapters and oligonucleotides for amplification and barcoding of sequences (Illumina R1 sequence (read 1 sequencing primer), 10x barcode, unique molecular identifier (UMI) and template switch oligo (TSO)). Subsequently, pooled GEMs of each sample were used for the construction of two libraries, the scRNA library containing the 5' gene expression profile and the single-cell TCR library comprising single-cell TRG and TRD sequences. Downstream processing and library construction, including clean-up steps, were performed according to the protocol provided by 10x Genomics. Agilent Bioanalyzer High Sensitivity chips were applied for quality control. To amplify γδ TCR chains, the primers were used for the enrichment of γδ TCR chains instead of primers for αβ TCR profiling (10x Genomics User Guide CG000086, Step 4, Target Enrichment from cDNA).    The scRNA-seq libraries were sequenced on the NextSeq 550 platform using read lengths of 26 base pairs (bp) read1 and 100 bp read2. The single-cell TCR libraries were sequenced by Illumina MiSeq.