GSM5702446: Thymus; Homo sapiens; RNA-Seq
Postnatal human thymus was obtained from children with congenital heart disease undergoing cardiac surgery at Rady Children's Hospital, San Diego, CA. Only patients who meet the inclusion criteria and sign informed consent, are included in the study. Thymus tissue was processed by mechanical dissociation into a single cell suspension, strained and viable lymphocytes were purified by Lymphoprep (StemCell) density gradient centrifugation before cryopreservation. For lung and peripheral blood samples, written, informed consent was obtained from all subjects from the Institutional Review Board of La Jolla Institute for Immunology and the Southampton and South West Hampshire Research Ethics Board. Newly diagnosed, untreated patients with non-small cell lung cancer were prospectively recruited once referred. Freshly resected tumor tissue and, where available, matched adjacent non-tumor lung tissue was obtained from patients with lung cancer following surgical resection. Tissues were macroscopically dissected and slowly frozen in 90% FBS (Thermo Fisher Scientific) and 10% DMSO (Sigma) for storage, until samples could be prepared. Cryopreserved non-tumor lung tissue was mechanically dissociated and enzymatically digested as previously described55. Briefly, lung tissue was minced with a scalpel and digested enzymatically with 0.15 WU/mL of D-Liberase (Roche) and 800 U/mL of DNase I (Sigma-Aldrich) for 15 min at 37°C. Then it was disaggregated into a single-cell suspension by passing it through a 70 µm strainer and rinsing with cold buffer (1× phosphate-buffered saline (PBS), 2 mM EDTA, 0.5% BSA). PBMCs were isolated using density gradient before cryopreservation. MAIT cells were sorted using MR1 tetramers. Single-cell libraries prepared as per the manufacturer's instructions (10x Genomics). Samples were processed using 10X v3 chemistry, as per the manufacturer's recommendations.