GSM6743985: 2cell_Nr5a2_CUT_RUN_rep2; Mus musculus; OTHER
To obtain mouse preimplantation embryos, 5-6-week-old C57BL/6N female mice were intraperitoneally injected pregnant mare's serum gonadotropin (PMSG; 5 IU) and human chorionic gonadotrophin (hCG; 5 IU) 44-48 hours after PMSG injection. After mated with PWK/PhJ males (Jackson Laboratory), preimplantation embryos were collected in M2 medium at following time points after hCG injection: 43 h (late 2-cell) and 68-70 h (8-cell). CUT&RUN was conducted following the published protocol (Skene et al., 2018). ESCs or embryos with zona pellucida removed were transferred into 0.2mL conventional, non-low-binding PCR tube (Axygen). The samples were resuspended by 60μL washing buffer (HEPES-KOH, pH = 7.5, 20mM; NaCl, 150mM; Spermidine, 0.5mM and with Roche complete protease inhibitor. Concanavalin-coated magnetic beads (Polyscience, 86057) 10-20μL for each sample were gently washed, resuspended by binding buffer (HEPES-KOH, pH = 7.9, 20mM; KCl, 10mM; CaCl2, 1mM; MnCl2, 1mM) and carefully added to the samples. The samples were then incubated at 23°C for 10 mins on Thermomixer (Eppendorf) at 400 rpm. The samples were then held at the magnetic stand to carefully aspirate buffer and were resuspended by 75μL antibody buffer (washing buffer plus 0.01% digitonin; EDTA, pH = 8.0, 2mM) with NR5A2 antibody (R&D, P-H2325-00) diluted at ratio of 1:100. The samples were then incubated at 4°C on Thermomixer for 3 hours at 400 rpm. After that, the samples were held at the magnetic stand and transfer to antibody buffer with rabbit anti-mouse IgG (Abcam, ab46540) diluted at ratio of 1:100. After that he samples were washed by 200μL dig washing buffer (washing buffer plus 0.01% digitonin) for once and 100μL for a second time. The samples were then resuspended by 75 μL dig washing buffer with pA-MNase (to a final concentration of 700ng/mL), and incubated at 4°C on Thermomixer for 1 hour at 400 rpm. The samples were washed by 200μL dig washing buffer twice at the magnetic stand. The samples were resuspended by 100μL dig washing buffer and balanced on ice for 2 mins. Targeted digestion was performed on ice by adding 2μL 100mM CaCl2 for 30 mins, and reaction was stopped by adding 100μL 2 × stop buffer (NaCl, 340mM; EDTA, pH = 8.0, 20mM; EGTA, pH = 8.0, 4mM; RNase A, 50μg/mL; glycogen, 100μg/mL) and fully vortexed. The samples were then incubated at 37°C of 30 mins for fragment releasing. The supernatants were purified by phenol-chloroform followed by ethanol purification.