GSM8772982: Stacc_GO_WT_2; Mus musculus; OTHER
Antibody and Hyperactive pAG-Tn5 Transposon (Vazyme, S614-01) complexes were prepared 20 minutes prior to the sample lysis step. One microlitre fully dissolved 5% digitonin (Sigma, D141) was added to 1 ml buffer 1 (10 mM Tris-HCl pH = 7.4, 150 mM NaCl, 0.5 mM spermidine, 1 × EDTA-free Roche complete protease inhibitor) to prepare DB-1 buffer. Seven microlitres of DB-1 buffer, 0.5 μl pAG–Tn5, and 0.5 μg antibody were added to a 200-μl low-binding tube and mixed by vortex. The mixture was incubated at 4 °C for 30 min. At the same time samples were transfer from the culture medium to wash buffer (DNase/RNase-free 0.01% BSA) with three washes and collected in DNA-lowing binding tubes (Each sample contains 50 oocytes) After removing the zona pellucida the samples were lysed in DB-1 buffer at 4℃ on the Thermo Mixer (TS100) at 500 rpm for 10 minutes, with vortexing every 2.5 minutes, three times. Thirty-five microlitres of DB-1 buffer, the pA/G–Tn5 mixture and 12.5 μl pre-warmed (37 °C) 5 × TTBL (Vazyme, TD502) were added into the resulting samples and then incubated in an Eppendorf Thermo mixer at 37 °C for 30 min at 400 rpm. Followed by the addition of 2 μl of 10% SDS and 40 ng of carrier RNA to the reacted samples in an Eppendorf Thermo Mixer at 55 °C for 30 minutes at 400 rpm. DNA was purified by 2x AMPure beads (Beckman, A63882). Then PCR was performed to amplify the libraries (Vazyme, TD601) using the following PCR conditions: 72 °C for 3 min; 98 °C for 30 s; thermocycling for 16 cycles at 98 °C for 15 s, 60 °C for 30 s and 72 °C for 3 min; followed by 72 °C for 5 min. After the PCR reaction, libraries were purified using 0.4×–1.7× AMPure beads (Beckman, A63882) for size selection and were subjected to next-generation sequencing.