GSM8807139: ESC-CTCF/WAPL-AID, E14, IAA treated 24 hours, ATAC-Seq; Mus musculus; ATAC-seq
The nuclei isolation was performed according to 10x Genomics guidelines. In brief, 500,000 trypsinized ESC were washed with PBS + 0.04% BSA and then lysed in 100ul Lysis Buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.01% Tween-20, 0.01% IGEPAL CA-630, 0.001% Digitonin, 1% BSA, 1mM DTT, 1 U/ul RNase inhibitor) for 5 minutes on ice. Immediately after, lysed cells were washed three times with 1ml Wash Buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 1% BSA, 0.1% Tween-20, 1mM DTT, 1 U/ul RNase inhibitor) and resuspended in 100ul Diluted Nuclei Buffer (1X Nuclei Buffer, 1mM DTT, 1 U/ul RNase inhibitor). Nuclei concentration was determined using LUNA Automated Cell Counter. Single-nucleus multiome libraries were prepared according to the 10x Genomics Multiome User Guide (CG000338). In short, nuclei were transposed and then partitioned with barcoded gel beads on a Chromium X controller. Transposed genomic DNA fragments and reverse-transcribed mRNA were single-cell barcoded, and libraries for ATAC and RNA-Seq were separated and processed in parallel. Sequencing libraries were prepared and quantified on an Agilent BioAnalyzer before being sequenced on an Illumina NovaSeq X Plus.