GSM5833524: Vd2 cells healthy; Homo sapiens; RNA-Seq
PBMCs were collected into Becton Dickinson Vacutainer® CPTTM Cell Preparation Tubes with Sodium Citrate. Cells were frozen in liquid nitrogen in a freezing medium containing RPMI, 10% DMSO and 12.5% HSA. Between 0.3 and 10 x 106 PBMCs were stained in PBS with LIVE/DEAD Zombie UV Fixable dye (Biolegend) for 20 min at room temperature. Human Fc block (Miltenyi Biotec) was then added for 10 minutes at 4ºC, followed by a staining with MR1‐5‐OP‐RU APC and CD1d-PBS-57 BV421 or MR1‐Ac‐6‐FP and CD1d unloaded tetramer (acquired from NIH tetramer core facility) for 40 minutes at room temperature. The tetramer staining was followed by surface antibody staining for 15 minutes at room temperature. In each batch of samples (n per batch ranged from 23 to 30) we included one or more healthy control samples stained with loaded and unloaded tetramer and in most of the batches we included a pooled patient sample stained with the unloaded tetramer. PBMCs from healthy samples were isolated using density centrifugation with Ficoll Paque®. PBMC samples were stained for flow cytometry and purified populations of MAIT and Vd2 cells from 5 patients and 5 healthy controls were collected using FACS sorting. The single-cell RNA-Seq of FACS-purifed cell suspensions was performed on Chromium instrument (10X genomics) following the user guide manual for 3′ v3.1. In brief, purified cells were washed once with PBS containing 1% bovine serum albumin (BSA) and resuspended in PBS containing 1% BSA to a final concentration of 700–1,300 cells per μl. The viability of cells was above 80%, as confirmed with 0.2% (w/v) Trypan Blue staining (Countess II). Cells were captured in droplets. Following reverse transcription and cell barcoding in droplets, emulsions were broken and cDNA purified using Dynabeads MyOne SILANE followed by PCR amplification per manual instruction. Between 6,000 to 30,000 cells were targeted for each sample. Samples were multiplexed together on one lane of 10X Chromium following cell hashing protocol. Final libraries were sequenced on Illumina NovaSeq S4 platform (R1 – 28 cycles, i7 – 8 cycles, R2 – 90 cycles). single cell RNA-seq