GSM8896475: adult iNKT cells, scTCR; Homo sapiens; OTHER
First, iNKT cells were enriched from PBMCs and CBMCs using MACS with anti-iNKT-Microbeads according to the manufacturer's guidelines, then stimulated with 2x10e5 allo-DCs in one well of a 24-well tissue culture plate in TCM containing αGalCer (100 nM) and IL-2 (200 IU/mL) for a 14-days. Growth factors and TCM were replenished every 2-3 days and iNKT cells were expanded into multiple wells if needed. Then, the freshly-expanded adult and cord-derived iNKT cells were stimulated by αGalCer-pulsed allogeneic dendritic cells for 24 hours and stained for CD3 and iNKTCR. Live iNKT cells were then sorted using BD FACSAria II Cell Sorter and submitted for single cells RNAseq analysis. The cells were processed through the 10X Genomics Chromium Next GEM Single Cell 5' V2 chemistry with TCR enrichment amplification following the manufacturer's protocol (CG000331). Briefly, cells were loaded onto a Chromium Next GEM Chip K (10X Genomics PN#1000286) at the recommended concentration to capture between 7,000 and 10,000 cells along with Single Cell 5' Gel Beads containing barcoded oligos. Using the Chromium Controller thousands of cells were partitioned into nanoliter-scale Gel Bead-in-Emulsion (GEM) droplets for single cell reactions. Within the droplets cells were lysed and the cellular RNA was barcoded using reverse transcriptase from the poly(dT) RT primers resulting in full-length cDNA from polyadenylated mRNA. The barcoded RNA is released, and pool of single cell libraries are amplified and cleaned up and then split. One portion is used for the full-length TCR are captured using TCR enrichment, and the second portion for the RNA library construction. The TCR region is selected using TCR primers using Chromium Single Cell Human TCR Amplification Kit (10X Genomics PN#1000252) and NGS libraries are constructed. The other portion of the amplified full length mRNA library is used to construct 5' RNA libraries using Chromium Next GEM Single Cell 5' Reagent Kit v2 (10X Genomics PN#1000263).