organism: Homo sapiens
description: Protocols: Samples were obtained from deceased transplant organ donors by the Collaborative Biorepository for Translational Medicine (CBTM) with informed consent from the donor families and approval from the National Research Ethics Services (NRES) Committee of East of England, Cambridge South (15/EE/0152). CBTM operates in accordance with UK Human Tissue Authority guidelines.  Tissue was collected from 5 donors from five lung locations including trachea, bronchi at the second/third generation, bronchi at the fourth generation, upper left lobe parenchyma and lower left lobe parenchyma. Following collection at the clinic, samples (range: 1–4 cm3) were immediately placed into cold Hypothermasol FRS32. Within 12 h after circulation ceased, samples were preserved in optimal cutting temperature (OCT) compound and frozen in isopentane at −60 °C for later nuclei isolation.  Our single nuclei isolation method from frozen tissue used 16 × 50 μm thick sections which were homogenized using a glass Dounce homogenizer (Sigma) in nuclei isolation buffer (NIM; 0.25 M sucrose, 0.005 M MgCl 2, 0.025 M KCl, 0.01 M Tris (buffer pH7.4), 0.001 M DTT and 0.1% Triton X-100) in the presence of Complete protease inhibitors (Roche) and RNAse inhibitors RNasin (Promega) 0.4 U μl−1 and SUPERase-In (Invitrogen) 0.2 U μl−1). Tissue was homogenized using ~15 strokes with pestle A (clearance 0.0028–0.0047 in.) and then pestle B (clearance 0.0008–0.0022 in.). Isolated nuclei were filtered through a 40 μM filter, collected at 500g and resuspended in 0.5 ml of storage buffer (PBS containing 4% BSA and RNasin (Promega) 0.2 U μl−1). Nuclei were incubated with 7-AAD (Ready Made Solution - Sigma-Aldrich SML1633-1ML) as recommended by the 10X protocol (CG000375 • Rev C) and purified from debris by FACs sorting, stained with Trypan blue and counted. Five thousand nuclei from five different samples were pooled and all 25,000 nuclei were further processed for 10x Chromium Next GEM Single Cell Multiome ATAC + Gene Expression according to the manufacturer's protocol (reference: CG000338). Nuclei suspensions were loaded with a targeted nuclei recovery of 15-16,000 droplets per reaction (to recover ~3,000 nuclei per sample). Samples were later demultiplexed by genotype. Quality control of cDNA and final libraries was carried out using Bioanalyzer High Sensitivity DNA Analysis (Agilent).