title: GSM8794427: ATAC_8C_Nr5a2_KD_rep2; Mus musculus; ATAC-seq
library_construction_protocol: Omni ATAC-seq was performed as described previously (Corces, M., Trevino, A., Hamilton, E. et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat Methods 14, 959–962 (2017)). Zona pellucida was removed by treatment with acidic Tyrode. Embryos were transferred into 1.5 ml of DNA LoBind Tube (Eppendorf) with 200 μl of PBS and were spun at 500×g at 4°C for 5 min to remove supernatant. 50 μl of ATAC-Resuspension buffer (RSB) (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2) containing 0.1% NP-40, 0.1% Tween-20 and 0.01% Digitonin was added into tube and incubated for 3 min on ice. After incubation, 200 μl of ATAC-RSB containing 0.1% Tween-20 was further added into the tube to wash out lysis. Nuclei were spun at 500×g at 4°C for 10 min to remove supernatant. Nuclei were incubated with 50 μl of transposition mixture (10 mM Tris-HCl (pH 7.6), 5 mM MgCl2, 10% Dimethyl formamide, 33% PBS, 0.1 % Tween-20, 0.01% Digitonin and 95 nM Tn5-adaptor complex) at 37°C for 30 min with 1000 rpm mixing. The samples were then deproteinized and further purified by phenol-chloroform and followed by ethanol precipitation. The DNA was amplified 50 µl of reaction with 1×NEBNext HF 2×PCR Master mix (NEB) and 0.25 µM barcoded primers using the following PCR program: 72 °C for 5 min; 98°C for 30 sec; 15 cycles of 98°C for 10 sec, 63°C for 30 sec and 72 °C for 1 min; final extension at 72 °C for 5 min and hold at 4°C. After PCR reaction, libraries were purified by 1.1×AMPure XP magnetic beads (Beckman Coulter) and were eluted in 20 μl of RNase-free water. Purified DNA libraries were sequenced on a NextSeq 500 (Illumina), Novaseq 6000 (Illumina), or AVITI (Element Biosciences) with paired-end reads.