GSM9067436: mESc 2i LIF 2 (scATAC-seq); Mus musculus; ATAC-seq
Nuclei were isolated from 4X105 mouse ES cells using 100 µL of cold lysis buffer (10 mM Tris-HCl (pH7.4), 10mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% IGEPAL CA-630, 0.01% Digitonin, 1% BSA, 1mM DTT, 1 U/µL RNase inhibitor). Cells were incubated for 5 min at 4°C, then resuspended in 1 mL chilled wash buffer (10 mM Tris-HCl (pH7.4), 10mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% BSA, 1mM DTT, 1 U/µL RNase inhibitor) and centrifuged at 500 rcf for 5 min at 4°C. Pellet was rinsed two more times with 1 mL wash buffer, for a total of 3 washes. The nuclei pellet was resuspended in 40 µL Diluted Nuclei Buffer (1X Nu- clei buffer (10X Genomics, PN-1000280), 1 mM DTT, 1 U/µL RNase inhibitor), and passed through a 40 µm Flowmi Cell Strainer (Merck) Library preparation was performed at the GenomEast platform at the Institut de Génétique et de Biologie Moléculaire et Cellulaire. Transposition was performed using the 10X Genomics kit (PN-1000280) and nuclei were loaded into the 10X Genomics Chromium Controller with a target capture of 6,000 single nuclei. Li- braries for the ATAC and 3′ mRNA were produced according to the Chromium Next GEM instructions (10X Genomics, Document CG000338 Rev F), and sequenced using the Illumina NextSeq 2000. Libraries were sequenced on an Illumina NextSeq 2000 sequencer as paired-end 28 + 85 base reads for 3′ mRNA, 50 base reads for ATAC. Image analysis and base calling were performed using RTA version 2.7.7 and BCL Convert version 3.8.4