title: TCR immune profiling of mononuclear cells from pooled:lung,spleen,bone marrow,mesenteric lymph node,thoracic lymph node
design: Tissue was obtained from deceased organ donors at the time of organ acquisition for clinical transplantation through an approved protocol and material transfer agreement via LiveOnNY, the organ procurement organization for the New York metropolitan area and through the Cambridge Biorepository for Translational Medicine. Each tissue was subjected to a tissue-specific protocol to maximize mononuclear cell (MNC) recovery and viability across a diversity of sites. A series of detailed protocols are available on protocols.io (https://dx.doi.org/10.17504/protocols.io.bwrjpd4n, https://dx.doi.org/10.17504/protocols.io.bwr9pd96, https://dx.doi.org/10.17504/protocols.io.bwsapeae, https://dx.doi.org/10.17504/protocols.io.bwq4pdyw, https://dx.doi.org/10.17504/protocols.io.bwq7pdzn, https://dx.doi.org/10.17504/protocols.io.bwr6pd9e, https://dx.doi.org/10.17504/protocols.io.bz4qp8vw). Each individual tissue single cell suspension (500,000 or 1,000,000 cells) was hash tagged to allow pooling of tissue samples. CITE-seq labeling used either a TotalSeq-C cocktail containing 137 protein targets (10x 5 libraries) or TotalSeq-A cocktail containing 277 protein targets (10x 3 libraries). For scRNA-seq experiments, single cells were loaded onto the channels of a Chromium chip (10x Genomics). cDNA synthesis, amplification, and sequencing libraries were generated using either the Single Cell 5 Reagent (v1 and v2) or 3 Reagent (v3) Kits. TCR and BCR paired VDJ libraries were prepared from samples made with the 5 Reagent kit. All libraries were sequenced on either an Illumina NextSeq 500/550 or NovaSeq 6000 instrument.