title: GSM7830549: MM_472, snATACseq; Homo sapiens; ATAC-seq
library_construction_protocol: Nuclei were isolated from frozen liver tissue using gentleMACS dissociation (Miltenyi). ~50-70 mg of frozen liver tissue was suspended in 1 ml of MACS dissociation buffer consisting of 5 mM CaCl2 (G-Biosciences, R040), 2 mM EDTA (Invitrogen, 15575-038), 1X protease inhibitor (Roche, 05-892-970-001), 3 mM MgAc (Grow Cells, MRGF-B40), 10 mM Tris-HCl pH 8 (Invitrogen, 15568-075), and 0.6 mM DTT (Sigma-Aldrich, D9779) in water (Corning, 46-000-CV) and placed on wet ice. Next, samples were homogenized using gentleMACS dissociator (Miltenyi) with gentleMACS M tubes (Miltenyi, 130-096-335) and the “protein_01” protocol. Suspension was filtered through a 30 µM CellTrics filter (Sysmex, 04-0042-2316). M tube and filters were washed with 1 ml of MACS dissociation buffer and combined with the suspension. Suspension was centrifuged in a swinging bucket centrifuge (Eppendorf, 5920R) at 500 rcf for 5 minutes (4°C, ramp speed 3/3). Supernatant was removed and pellet was resuspended in 1 mL Nuclear Permeabilization Buffer (1X PBS, 5% Bovine Serum Albumin, 0.2% IGEPAL CA-630 (Sigma), 1 mM DTT, 1X complete Protease inhibitor). Nuclei were rotated at 4 °C for 5 minutes before being pelleted again with a swinging bucket centrifuge (500 x g, 5 min, 4°C; 5920R, Eppendorf). After centrifugation, permeabilized nuclei were resuspended in 500 μL high salt tagmentation buffer (36.3 mM Tris-acetate (pH = 7.8), 72.6 mM potassium-acetate, 11 mM Mg-acetate, 17.6% DMF) and counted using a hemocytometer. For single nucleus RNA-seq, 75,000 nuclei were sorted using a SH800 sorter (Sony) into 50 ul of collection buffer consisting of 1 U/ul RNase inhibitor in 5% BSA; the FACS gating strategy sorted based on particle size and DRAQ7 fluorescence. Sorted nuclei were then centrifuged at 1000 rcf for 15 minutes (4°C, run speed 3/3) and supernatant was removed. For single nucleus ATAC-seq, unsorted nuclei were used as input for single nucleus ATAC-seq using combinatorial barcoding For single nucleus ATAC-seq, unsorted nuclei were used as input for single nucleus ATAC-seq using combinatorial barcoding. Approximately 15,000 nuclei were loaded onto the Chromium controller (10x Genomics) for downstream single-nucleus RNA-seq processing with the Single Cell 3' v3.1 GEM generation kit (10x Genomics). single nucleus ATAC-seq, single nucleus RNA-seq