GSM5861167: Cdc14ab KO time 5 [ATAC-Seq]; Mus musculus; ATAC-seq
ESCs in LIF-containing media (T0) or after 5 days (T5) in neural differentiation media were washed twice with PBS, trypsinized and counted. Dead cells were removed with Dead Cell Removal kit (Miltenyi Biotec). Cells were counted again and 1 million cells were centrifuged at 300 g 10 min at 4oC in PBS 0.04% BSA. Cells were treated with 0.1 U/l of DNAse I (Invitrogen) 5 min in ice. Cells were washed in PBS 0.04% BSA and centrifuged 10 min at 300 g twice. Cells were incubated 2 min with 100 l Lysis Buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% NP40, 0.01% Digitonin, 1% BSA, 1 mM DTT, 1 U/l RNase inhibitor) in ice for nuclei extraction. After incubation, nuclei were washed in 1 ml of Wash Buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% BSA, 1 mM DTT, 1 U/l RNase inhibitor) and centrifuged 5 min at 500 g. Washing was repeated two more times and nuclei were filtered in 40 m Flowmi Cell Strainer, counted and assessed for integrity. Cell nuclei were loaded onto a Chromium Next GEM Chip J (10x Genomics) for a target recovery of ~5000 nuclei per condition, and processed into a 10x Chromium Controller as described in the manufacturer's protocol (Chromium Next GEM Single Cell Multiome ATAC + Gene Expression, CG000338 Rev A). Generation of gel beads in emulsion (GEMs), barcoding, pre-amplification PCR and ATAC library construction were all performed as recommended by the manufacturer. snATAC