GSM5203367: SAMP-1144; Homo sapiens; ATAC-seq
Approximately 1,000 islet equivalents (~1,000 cells per IEQ) were resuspended in 1 ml nuclei permeabilization buffer (10mM Tris-HCL (pH 7.5), 10mM NaCl, 3mM MgCl2, 0.1% Tween-20 (Sigma), 0.1% IGEPAL-CA630 (Sigma) and 0.01% Digitonin (Promega) in water) and homogenized using 1ml glass dounce homogenizer with a tight-fitting pestle for 15 strokes. Homogenized islets were incubated for 10 min at 4°C and filtered with 30 µm filter (CellTrics). Nuclei were pelleted with a swinging bucket centrifuge (500 x g, 5 min, 4°C; 5920R, Eppendorf) and washed twice with Wash buffer (10mM Tris-HCL (pH 7.5), 10mM NaCl, 3mM MgCl2, 0.1% Tween-20, and 1% BSA (Proliant 7500804) in molecular biology-grade water). Nuclei were pelleted and resuspended in 30 µL of 1X Nuclei Buffer (10x Genomics). Nuclei were counted using a hemocytometer, and 15,360 nuclei were used for tagmentation. Single nucleus ATAC-seq libraries were generated using the (Chromium Chip E Single Cell ATAC kit (10x Genomics, 1000086) and indexes (Chromium i7 Multiplex Kit N, Set A, 10x Genomics, 1000084) following manufacturer instructions. Final libraries were quantified using a Qubit fluorimeter (Life technologies) and the nucleosomal pattern was verified using a Tapestation (High Sensitivity D1000, Agilent). Libraries were sequenced on NextSeq 500 and HiSeq 4000 sequencers (Illumina) with following read lengths: 50 + 8 + 16 + 50 (Read1 + Index1 + Index2 + Read2).