GSM5451971: PNT2_TCR; Homo sapiens; OTHER
Cell suspensions from fetal thymus and pediatric thymus samples were obtained as previously described (Tieppo et al. JEM 2020) and cryopreserved in liquid nitrogen with freezing media (10%DMSO, 90%FBS). When samples were used, cells were thawed and washed twice with RPMI 1640 (Gibco) complete medium, labeled with Zombie NIR dye (BioLegend), and then subsequently stained for cell surface Ags with Abs directed against CD3 (clone UCHT1; BD Biosciences), TCR-γδ (11F2; BD Biosciences), TCR-Vγ9 (IMMU360; Beckman Coulter), TCR-Vδ2 (IMMU389; Beckman Coulter). For the sc experiment, CD3+ γδTCR+ were sorted for “γδ” thymocytes (mean purity 98% of living cells) on a FACS Aria III (BD Biosciences). Libraries for sc RNA and TCR sequencing were generated from 1.5-2*104 FACS-sorted γδ thymocytes using the Chromium Single Cell 5' Library Gel Bead and Construction kit as well as Chromium Single Cell V(D)J Enrichment Kit (10x Genomics, CA, USA) according to the user guidelines (v1 Chemistry, Single Cell V(D)J protocol number CG000086). Single-cell TCR and gene expression libraries were generated according to Chromium Single Cell V(D)J protocol (10x Genomics). 2μL of cDNA amplified and purified from GEMs (“Gel bead in EMulsion” droplets) were used to amplify γδTCR CDR3 sequences. Custom primers specific for TRDC and TRGC constant gene segments were designed for this purpose. The remaining of cDNA was used for gene expression library construction according to 10X Genomics protocol instructions. Agilent Bioanalyzer High Sensitivity DNA chips were used to check quality control read-outs of scRNA-seq and scTCR-seq libraries using a Bioanalyzer 2100 machine (Agilent Technologies). The scRNA-seq libraries were sequenced on the Illumina NovaSeq 6000 platfotm from BRIGHTcore (Brussels University Alliance).