GSM9310103: Gata1_8C_CUTnRUN_rep1; Mus musculus; OTHER
CUT&RUN was performed according to the published protocol (Hayashi and Inoue, 2023) with a few modifications. 8-cell embryos were treated with acidic Tyrode to remove the zona pellucida, and washed through 0.15% BSA in PBS on a microplate (NuncTM MicrowellTM Minitrays). Embryos were incubated in antibody buffer (20 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 0.5 mM Spermidine, 0.02% Digitonin, 2 mM EDTA and 1x Protease inhibitor cocktail (Roche) with primary antibody (1:100 dilution) overnight at 4°C on a nutator. Embryos were washed three times through antibody buffer and further incubated with secondary antibody (1:100 dilution) for 1 hour at room temperature. After incubation, embryos were washed three times through cold Dig-Wash buffer (20 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 0.5 mM Spermidine, 0.02% Digitonin, and 1x Protease inhibitor cocktail), followed by incubation in Dig-Wash buffer for 20 min at room temperature. Embryos were further incubated with cold Dig-Wash buffer containing homemade pAG-MNase (500 ng/µl) for 1.5 h on ice with gentle nutation. Embryos were washed twice through cold Dig-Wash buffer and incubated for 20 min at room temperature with gentle nutation. Embryos were transferred into a 1.5 mL tube containing 50 µL of the wash buffer and then captured by Bio-Mag Plus Concanavalin A-coated beads. After removing the supernatant, 180 µL of cold Dig-wash buffer was added, and MNase reaction was carried out in the presence of 2 mM CaCl2 for 20 min on ice. The reaction was stopped by adding 20 µL of 10XSTOP buffer (1700 mM NaCl, 500 mM EDTA, 22 mM EGTA, 0.02% digitonin, 250 μg/mL RNase A, 500 μg/mL glycogen). The protein–DNA complexes were then released by 20 min incubation at 37°C with shaking at 1000 rpm. The supernatant was transferred into a new 1.5 ml of DNA LoBind Tube (Eppendorf) and then deproteinized. The DNA was purified by phenol-chloroform extraction and followed by ethanol precipitation. DNA libraries were prepared using a NEBNext Ultra II DNA library preparation kit for Illumina (New England Biolabs) according to the manufacturer's instructions Purified DNA libraries were sequenced on a AVITI (Element Biosciences) with paired-end reads.