Coupled single-cell RNA, TCR, and barcoded pMHC multimer sequencing, from virus-specific PBMC T-cells
Coupled single-cell RNA, TCR, and barcoded pMHC multimer sequencing, from virus-specific PBMC T-cells
Whole blood from 27 anonymous blood donors were obtained at the central blood bank at Rigshospitalet, Copenhagen, and enriched for PBMCs using density centrifugation in Leucosep tubes before cryopreservation at -150°C in FCS plus 10% DMSO PBMCs from 21 hepatitis patients were collected and cryopreserved under protocol ID #1999-P004983 “Cell Mediated Immunity in Viral hepatitis ~15,000 cells were loaded (based on 55% recovery from 27,500 sorted cells) onto a Chromium Controller to yield a maximum of 9,000 cells with an intermediate/high doublet rate (6.9%). We utilized the 10x Genomics 5' v2 chemistry protocol supplied by 10x genomics allowing the cell barcode to be appended at the 5'-end of transcripts during capture. See https://elifesciences.org/articles/81810#s4 for further details. Downstream processing of mRNA and DNA barcodes is performed according to the manufacturer's instructions (Chromium Next GEM Single Cell 5' Reagent Kits v2 [Dual Index], with the Feature Barcode technology for Cell Surface Protein & Immune Receptor Mapping) (10x Genomics, USA). Targeted amplification was performed for 13 cycles and the products were separated according to size into <400 bp (DNA barcode-tags) and >400 bp (the TCR sequences) using 0.6× SPRIselect beads (Beckman Coulter, B23318).