Journal: Nature Communications

Article Title: High throughput pMHC-I tetramer library production using chaperone-mediated peptide exchange

doi: 10.1038/s41467-020-15710-1

Figure Lengend Snippet: Fluorophore-labeled, empty MHC-I/TAPBPR multimers are loaded with peptides of interest on a 96-well plate format and individually barcoded with DNA oligos designed for 10x Genomics and Illumina compatibility. TAPBPR and excess peptide, along with free oligo, are removed by centrifugation and the multimers are pooled together. A single patient sample can be stained with the pooled multimer library, and collected by fluorescence-activated cell sorting. Tetramer associated oligos and cellular mRNA from individual cells are then barcoded using 10x Genomics gel beads, followed by cDNA synthesis, library preparation and library sequencing. This workflow enables the transcriptome and paired αβ TCR sequences to be linked with pMHC specificities in a single experiment.

Article Snippet: Post sorting, samples were prepped for the 10X Genomics 5 P V(D)J kit workflow, and processed according to the ECCITE-seq protocol , with the modifications below (Supplementary Fig. a).

Techniques: Labeling, Centrifugation, Staining, Fluorescence, FACS, cDNA Synthesis, Sequencing