GSM5885672: scATAC-Seq day 5; Homo sapiens; ATAC-seq
Epicardioids were dissociated with papain as described in methods of Meier et al. (2021), filtered through 40 μm filter, washed with 5 mL 1% BSA (Gibco; 15260037) in PBS-/-, centrifuged for 3 min at 200 g, and resuspended in 0.04% BSA in PBS for counting. Nuclei isolation was performed following the “Nuclei Isolation for Single Cell ATAC Sequencing demonstrated protocol” (10x Genomics). Briefly, ~500,000 cells from each sample were transferred to a 1.5-ml microcentrifuge tube and centrifuged at 300×g for 5 min at 4 °C. The supernatant was removed without disrupting the cell pellet, and 100 μl of chilled Lysis Buffer (10 mM Tris-HCl (pH 7.4); 10 mM NaCl; 3 mM MgCl2; 0.1% Tween-20; 0.01% Nonidet P40 Substitute; 0.01% Digitonin and 1% BSA) was added and pipette-mixed 10 times. Samples were then incubated on ice for 30-120 seconds (depending on sample quality and day; optimal incubation time was optimized in advance). Following lysis, 1 mL of chilled Wash Buffer (10 mM Tris-HCl (pH 7.4); 10 mM NaCl; 3 mM MgCl2; 0.1% Tween-20 and 1% BSA) was added and pipette-mixed. Nuclei were centrifuged at 500×g for 5 min at 4 °C, and the supernatant removed without disrupting the pellet, and nuclei were resuspended into the appropriate volume of chilled Diluted Nuclei Buffer (10x Genomics) in order to achieve a nuclei concentration suitable for a target nuclei recovery of 8000. 8 000 cells per sample were processed using Chromium Next Single Cell ATAC Library & Gel Bead Kit v1.1 (1000175, 10x Genomics) Chromium Single Cell H Chip Kit (1000161, 10x Genomics) and Chromium  SIngle Index Kit N, Set A (PN-1000212; 10x Genomics) to generate Gel Bead-In-EMulsions (GEMs) and single cell ATAC sequencing libraries. Libraries were pooled and sequenced using the NovaSeq (Ilumina; S1 flow cell)  with 150bp paired end reads with 50 cycles for read1 and read2 8 cycles for i7 and 16 cycles for i5, and with a read depth of at least 25-30 000 pair reads per cell.