Instrument: Illumina NovaSeq 6000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Blood and MLR Samples: RM PBMCs were isolated from whole blood by Ficoll gradient centrifugation, and then used for MLR assays either immediately, or after liquid nitrogen cryopreservation in 10% dimethyl sulfoxide (DMSO)/90% fetal bovine serum (FBS). Frozen samples were thawed using RPMI-1640 media prior to resuspension in X-vivo-15 medium (BioWhitaker) supplemented with with 10% FBS (Irvine Scientific) for MLR incubation. At the time of the MLR, stimulator PBMCs were irradiated with 3500 cGy of (137Cs) radiation. Responder PBMCs were stained with cell trace violet (CTV, Invitrogen) as per manufacturer's instructions. 2× 105 T cell-enriched responder PBMCs along with an equal number of stimulator PBMCs were added to each well in a 96-well plate (Corning) in X-vivo-15 medium supplemented with 10% FBS and incubated at 37°C for a total of 5 days. Cell culture media change was performed on day 3 of the culture. At the end of 5 days, cells were stained with an extracellular antibody for CD3 (clone SP34-2), CD20 (clone 2H7) and CD14 (clone M5E2, all antibodies from BD Bioscience) for 20min at 4°C, and high-, medium- and non-proliferating CD3+ T cells (identified based on the dilution of CTV) were sorted and processed for single cell sequencing using the Chromium Next GEM Single Cell 5' Reagent Kit with optimized RM primers as described below. GvHD Organ Samples: Viable LIVE/DEAD Violet-negative CD45-positive cells were sorted from two out of four GVHD-derived samples into host and donor fractions, based on MAMU-A001 expression. 10x Genomics Single Cell 5' Reagent Kit for Single-cell RNA-Seq and with custom RM primers for single-cell TCR-Seq