PMC3778833_01
Flow cytometry and generation of the final amplicon library.. (a) Sorting gate. The initial gates were set on lymphocytes based on side scatter (SSC) and forward scatter (FSC), from which the CD3 T cell gate (left panel) was set. Within the CD3 gate, we firstly gated for CD3+CD25+CD127-/lo natural Treg ('Treg') cell population (middle panel) and the remaining CD3+ T cells were divided into CD3+CD4+ ('CD4+') and CD3+CD4- ('CD4-') conventional T cells (right panel). (b) Gel appearance of the final purified amplicon library. RNA was purified from sorted cells representing the 12 samples (corresponding to three T cell subpopulations at the four time points) and TRB V-D-J transcripts were amplified, independently, through 5'RACE and PCR and barcodes incorporated in a second PCR. The products were purified and an equal amount (100 ng) of cDNA from each of the 12 reactions was pooled. The final amplicon library appeared as a band between 550 and 650 bp.
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T cell subpopulations: regulatory T cells ('Treg', with a phenotype CD3+CD25+CD127-/lo) and conventional T cells CD3+CD4+ ('CD4+') and CD3+CD4- ('CD4-') using FACSAria (BD Biosciences) (Fig. 1a). Treg cells, which represent a minor subpopulation (~5% ) within circulating T cells (or ~2% of PBMC) were included in the analysis to evaluate if the technique works for both abundant T cell subpopulations (e.g., CD4+) as well as small subpopulations. Finally, an equal amount (100 ng) of cDNA from each of the 12 libraries was pooled to obtain the final amplicon library, which represents the complete collection of TRBV transcripts sampled from this donor (Fig. 1b).