GSM6190657
PBMCs were obtained from HIV-1 infected, aviraemic individuals under ART by leukapheresis and stored on liquid nitrogen. CD4+ CD45RA- cells were isolated by magnetic-activated cell sorting. Cells were subsequently incubated with Fc blocking reagent, and stained with a combination of anti-CD3 and/or anti-CD4, anti-TRBC1, and one or a combination of various anti-TRBV antibodies. Cells were sorted for CD3 and/or CD4, a specific TRBC domain and/or a specific TRBV domain or a subset of various TRBV domains. Library was performed according to the manufacter's instructions (single cell 5' and V(D)J Enrichment v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.