GSM6005264: SAMP-1150_ATAC; Homo sapiens; ATAC-seq
Islets were resuspended in 1 mL wash buffer (10mM Tris-HCL (pH 7.4), 10mM NaCl, 3mM MgCl2, 0.1% Tween-20 (Sigma), 1% fatty acid-free BSA (Proliant, 68700), 1 mM DTT (Sigma), 1x protease inhibitors (Thermo Fisher Scientific, PIA32965), 1U/µl RNAsin (Promega, N2515) in molecular biology-grade water) and homogenized using 1 mL glass dounce homogenizer with a tight-fitting pestle (Wheaton, EF24835AA) for 10-20 strokes until the solution was homogeneous. Homogenized islets were filtered with 30 µm filter (CellTrics, Sysmex) and pelleted with a swinging bucket centrifuge (500 x g, 5 min, 4°C; 5920R, Eppendorf). Nuclei were resuspended in 400 µL of sort buffer (1% fatty acid-free BSA, 1x protease inhibitors (Thermo Fisher Scientific, PIA32965), 1U/µl RNAsin (Promega, N2515) in PBS) and stained with 7-AAD (1 µM; Thermo Fisher Scientific, A1310). 120,000 nuclei were sorted using an SH800 sorter (Sony) into 87.5 μl of collection buffer (1U/µl RNAsin (Promega, N2515), 5% fatty acid-free BSA (Proliant, 68700) in PBS). Nuclei suspension was mixed in a ratio of 4:1 with 5x permeabilization buffer (50 mM Tris-HCL (pH 7.4), 50 mM NaCl, 15 mM MgCl2, 0.5% Tween-20 (Sigma),  0.5% IGEPAL-CA630 (Sigma), 0.05% Digitonin (Promega), 5% fatty acid-free BSA (Proliant, 68700), 5 mM DTT (Sigma), 5x protease inhibitors (Thermo Fisher Scientific, PIA32965), 1U/µl RNAsin (Promega, N2515) in molecular biology-grade water) and incubated on ice for 1 min before pelleting with a swinging-bucket centrifuge (500 x g, 5 min, 4°C; 5920R, Eppendorf). Supernatant was gently removed and ~50 µl were left behind to increase nuclei recovery. 650 µl of wash buffer (10mM Tris-HCL (pH 7.4), 10mM NaCl, 3mM MgCl2, 0.1% Tween-20 (Sigma), 1% fatty acid-free BSA (Proliant, 68700), 1 mM DTT (Sigma), 1x protease inhibitors (Thermo Fisher Scientific, PIA32965), 1U/µl RNAsin (Promega, N2515) in molecular biology-grade water) were added without disturbing the pellet and nuclei were pelleted with a swinging bucket centrifuge (500 x g, 5 min, 4°C; 5920R, Eppendorf). Supernatant was gently removed without disturbing the pellet and leaving ~2-3 µl behind. 7-10 µl of 1x Nuclei Buffer (10x Genomics) was added and nuclei gently resuspended. Nuclei were counted using a hemocytometer, and 16,550-18,000 nuclei were used as input for tagmentation. Single Cell Multiome ATAC + Gene Expression libraries were generated following manufacturer instructions (Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle, 1000283; Chromium Next GEM Chip J Single Cell, 1000234; Dual Index Kit TT Set A, 1000215; Single Index Kit N Set A, 1000212; 10x Genomics) with these PCR cycles: 7 cycles for ATAC index PCR, 7 cycles for cDNA amplification, 13-16 cycles for RNA index PCR. Final libraries were quantified using a Qubit fluorimeter (Life Technologies) and the size distribution was checked using a Tapestation (High Sensitivity D1000, Agilent). Libraries were sequenced on NextSeq 500 and NovaSeq 6000 sequencers (Illumina) with following read lengths (Read1 + Index1 + Index2 + Read2): ATAC (NovaSeq 6000) 50 + 8 + 24 + 50; ATAC (NextSeq 500 with custom recipe) 28 + 10 + 10 + 90;  RNA (NextSeq 500, NovaSeq 6000): 50 + 8 + 16 + 50.