GSM6255601: D511 GEX spleen; Homo sapiens; RNA-Seq
Tissue samples were maintained in cold saline or media and transported to the laboratory within 2-4 hrs of organ procurement. Tissue processing protocols were adapted from protocols previously described [Poon et al, Cell Reports, 2021; Carpenter et al, Am. J. Transplant, 2018; Senda et al, Mucuosal Immunol, 2019; Dogra et al, Cell, 2020; Poon et al, Sci Immunol, 2021], and processed in a timely manner, resulting in high yields of live leukocytes. Briefly, mononuclear cells were isolated from the blood and BM samples by density centrifugation using Lymphocyte Separation Medium (Corning, cat# 25-072-CI) or Ficoll-Paque PLUS (GE, cat# 17-1440-03). Spleen was processed using mechanical dissociation, followed by density centrifugation, as above. Lung, jejunum, and lymph node samples were processed using mechanical and enzymatic digestion, followed by density centrifugation, as previously described [Kumar et al, Immunity, 2018; Poon et al, Cell Reports, 2021; Senda et al, Mucosal Immunol, 2019; Poon et al, Sci Immunol, 2021; Wells et al, protocols.io, 2021]. Skin samples were carefully washed using cell culture medium, cleaned by scraping the subcutaneous fat with a scalpel, and washed with cell culture medium again. Then, skin samples were cut into pieces of approximately 4mm in width and digested overnight at 37ºC using the Human Skin Dissociation Kit (Miltenyi). The following day, mononuclear cells were washed and isolated. Using the Chromium Next GEM single cell 5' Reagent kit v2 (10X Genomics), sorted T cells from each tissue site were loaded onto separate lanes of the Next GEM Chromium Controller (10X Genomics) for encapsulation (target recovery of 5,000 cells for each sample). Single cell libraries were constructed using manufacturer's protocols. TCR sequencing libraries for TCRαβ were prepared with the V(D)J enrichment kit from 10X Genomics, following manufacturer's protocols.