GSM5742999: Naive_primed_D30ASECRiAV_10XscATACseq; Homo sapiens; ATAC-seq
Cells were washed with Phosphate-buffered Saline (PBS), dissociated from culture dishes by Accutase (7 min. at 37°C) in hypoxic condition for naive hPSCs and TSCs and in normoxic condition for primed hPSCs, and finally diluted with DMEM. Single cell suspensions were filtered through a 40µm cell strainer, centrifuged at 200 rcf for 5 min., resuspended in PBS with 0.04% Bovine Serum Albumin (BSA), and counted with a Luna-FL automated Fluorescence Cell Counter (Logos Biosystems). 100 000 – 1 000 000 cells were added to a 2-ml microcentrifuge tube and were centrifuged (300 rcf, 5min at 4°C). Supernatant was removed without disrupting the cell pellet and 100µl chilled lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% Nonidet P40 Substitute, 0.01% digitonin and 1% BSA) was added and mixed by pipetting 10 times. The microcentrifuge tube was incubated on ice for 4 min and then 1ml 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 mixed by pipetting 5 times. Nuclei were centrifuged (500 rcf, 5 min at 4°C) and the supernatant was removed without disrupting the nuclei pellet. Nuclei were resuspended in a chilled Diluted Nuclei Buffer (10x Genomics; 2000153) at 610-1540 nuclei per µl based on the starting number of cells. The nuclei concentration was determined using a NucleoCounter NC-100 (Chemometec). Nuclei were mixed using the following ratio (H9 primed: Sigma primed: H9 naive: Sigma naive: Sigma TSCs = 1:1:1:1:2) and were immediately used to generate scATAC-seq libraries as described in the methods below. scATAC-seq libraries were prepared according to the Chromium Single Cell ATAC Reagent Kits User Guide (10x user guide; CG000168, Revision D). Nuclei were loaded onto the 10x Chromium Single Cell Platform (10x Genomics) at a concentration targeting 2000 nuclei, according to the manufacturer's protocol. Nuclei transposition, generation of gel beads in emulsion (GEMs), barcoding, GEM cleanup, and library construction were performed with the Chromium Single Cell ATAC Reagent Kits (v1 Chemistry) according to the manufacturer's protocol. Library Quality control and quantification was assessed using the Tapestation (Agilent) and Qubit 2.0 (ThermoFisher Scientific). The Library was sequenced on a Nextseq500 (Illumina) instrument using the MID output kit (Illumina) (20024904).