10X Genomics Immune Profiling of homo sapiens: Pre and post vaccination. Samples are multiplexed using Cell hashing and must be demultiplexed after running through CellRanger software
Cryopreserved PBMC were thawed and rested for 3 hours. Post rest, cells were incubated with 1 g/ml anti-CD40 antibody (Miltenyi) and anti-human CD28/49d (BD Biosciences) at a concentration of 5106 cells/ml.  A minimum of one million cells were plated for each stimulation condition. Those from the BCG group were stimulated with gamma-irradiated M.tb H37Rv whole cell lysate (BEI resources, cat NR-14822, final concentration of 30 g/ml), or PBS (Gibco/ThermoFisher; negative control), whereas those from the H4:IC31 group were stimulated with the Ag85B/TB10.4 combined peptide pool (concentration 1 g/ml of each peptide, Biosynthesis Inc.) or DMSO (0.5%, Sigma Aldrich; negative control), and incubated at 37C/5% CO2 for 16 hours. At the end of the stimulation period, cells were washed and stained with LIVE/DEAD Aqua viability dye and then washed and sequentially stained with the oligo labeled MR1-5-OP-RU  for 15 minutes, and the tained with a pool of 108 oligo-labeled antibodies (TotalseqC, Biolegend), and a pool of fluorochrome labeled antibodies (CD3, CD4, CD8, CD137, CD69, CD154) and each stimulation condition was also stained with a pre-specified cell Hashtag ((Human, TotalseqC, Biolegend, see supplementary files:HTO_demultipex.csv, 602_Feature Barcode.csv). Cells were sorted on the FACSAria II SORP (BD Biosciences) equipped with DIVA 8.0.1 software. The following populations were sorted into 2 ml LoBind tubes (Eppendorf) containing 20 l FcX CSB 200 U/ml RNasin:  CD3+CD4+CD154+CD69+ and CD3+CD8+CD69+CD137+ T cells (antigen-reactive CD4+ and CD8+ T cells), non-antigen-reactive T cells (CD3+CD4+ but not CD154+, or CD3+CD8+ but not CD137+), and CD3- cells from each stimulation condition. Captured cells were pooled according to a predetermined pooling and hashtag schema (see HTO_demultipex.csv supplementary file) and then processed using the 10X Genomics 5 immune profiling with VDJ and feature barcoding version 1.1 kits. Gene expression, Feature barcode (including hashtags (HTO)), and VDJ (TCR /) libraries that were indexed and sequenced using the Illumina Novaseq 6000 (Illumina) sequencing platform using S4 and S2 200 cycle flow cells per the manufacturers instructions.