GSM6123273: MM290_D14; Homo sapiens; ATAC-seq
At D11, D14, D21, D32 and D39 of differentiation, approximately 1,000 aggregates (~1,500 cells per aggregate) were collected 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 for 15 strokes with a tight-fitting pestle. To extract nuclei, homogenized aggregates 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 Next GEM Single Cell ATAC Library & Gel Bead Kit v1.0 (10x Genomics, 1000175), Chromium Next GEM Chip H Single Cell Kit (1000161) and Single Index Kit N Set A (1000212)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 or NovaSeq 6000 sequencers (Illumina). Reads were trimmed afterwards to fit into corresponding analysis pipeline. Single nucleus ATAC-seq Chromium Next GEM Single Cell ATAC Library & Gel Bead Kit v1.0.