title: GSM6459713: PDSN09, snATAC-seq; Homo sapiens; ATAC-seq
library_construction_protocol: About 15 mg of tissue was placed in an extra-thick tissue processing tube (Covaris, 520140) while kept in liquid nitrogen, and repeatedly hammered to produce frozen tissue powder. The frozen tissue was transferred to lysis buffer of 0.2% Triton X-100, protease inhibitor (Roche, 04-693-159-001), 1 mM DTT (Sigma-Aldrich, D9779), 0.2 U/µL RNAsin (Promega, N211B), and 2% BSA (Sigma-Aldrich, SRE0036) in PBS. Sample was pipetted 10 times, rotated for 5 min at 4°C, and centrifuged at 500g for 5 min. The pellet was resuspended in sorting buffer of 1 mM EDTA, 0.2 U/µL RNAsin, and 2% BSA in PBS, and passed through a 30 µm strainer (Sysmex, 04-0042-2316) to remove excessive debris. The nuclei were stained with DRAQ7 (1:100, Cell Signaling, 7406) for snRNA-seq, and with DAPI (10 ug/mL, Sigma, 32670) for snATAC-seq. Between 100,000 and 150,000 nuclei were sorted using a MoFlo Astrios EQ sorter (Beckman coulter, laser/filter) into a collection tube with 1 U/ µL RNAsin and 5% BSA in PBS. Sorted nuclei were centrifuged at 1000 g for 15 min at 4°C, and supernatant was removed. Nuclei were resuspended in resuspension buffer (0.2 U/µL RNAsin and 1% BSA in PBS) and counted using a cell counter (Cellometer Auto 2000, Nexcelom Biosicence) with AO/PI staining. The isolated nuclei were subject to permeabilization in a lysis buffer with Tris-HCl (pH8.0), 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% Nonidet P40 Substitute (Sigma-Aldrich, 74385), 0.01% digitonin (Thermo Fisher Scientific, BN2006), and 1% BSA. snATAC-seq libraries were generated using the Chromium Single Cell ATAC Library & Gel Bead Kit v1 (10x Genomics).