title: GSM8794342: Nr5a2_4C_rep2; Mus musculus; OTHER
library_construction_protocol: CUT&Tag was performed as described previously with few modifications (Kaya-Okur, H.S., Wu, S.J., Codomo, C.A. et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun 10, 1930 (2019)). Embryos with intact zona pellucida were incubated with ice-cold extraction buffer (25 mM HEPES-NaOH (pH 7.4), 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose and 0.5% Triton X-100) on ice for 6-7 min and were washed three times through with ice-cold extraction buffer without TritonX-100. Pre-extracted embryos were immediately lightly fixed by Dulbecco's phosphate- buffered saline (DPBS) with 0.1% formaldehyde for 2 min at room temperature. Embryos were incubated in antibody buffer (20 mM HEPES-NaOH (pH7.5), 150 mM NaCl, 0.5 mM Spermidine, 0.1% BSA, 2 mM EDTA and 1x Protease inhibitor cocktail (Roche) with primary antibody (1:100 dilution) overnight at 4°C on nutator. Embryos were washed three times through antibody buffer and further incubated with secondary antibody (1:100 dilution) for 1.5 hours at room temperature. Cells were washed three times through Wash buffer (20 mM HEPES-NaOH (pH7.5), 150 mM NaCl, 0.5 mM Spermidine and 1x Protease inhibitor cocktail (Roche)) and further incubated with homemade pA-Tn5 or pAG-Tn5 adaptor complex (1:250 dilution) for 1.5 hours at room temperature. Embryos were washed three times through Wash-300 buffer (20 mM HEPES-NaOH (pH7.5), 300 mM NaCl, 0.5 mM Spermidine and 1x Protease inhibitor cocktail (Roche)). Embryos were transferred into 1.5 ml of DNA LoBind Tube (Eppendorf) with 200 µl of Tagmentation buffer (10 mM MgCl2 in Wash-300) and incubated at 37°C for 1 hour. The samples were then deproteinized and reverse-crosslinked. The DNA was purified by phenol-chloroform and followed by ethanol precipitation. The DNA was amplified 50 µl of reaction with 1×Q5 High-Fidelity 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 and 63°C for 10 sec; final extension at 72 °C for 1 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.