GSM7776075: K27-zygote-3.3con [Cut&Tag]; Mus musculus; OTHER
Embryos were incubated in 50 µL antibody buffer; 0.5 μg antibody was added and the mixture was incubated at 4℃ overnight. After washing twice with dig-wash buffer, 50 µL dig-wash buffer with 0.2 µg secondary antibody was added and the mixture was incubated at room temperature for 1 h. After washing twice with 100 µL dig-wash buffer, 1 µL pG-Tn5 and 49 µL dig-300 buffer were added, and the samples were incubated at room temperature for 1 h. Next, the samples were washed twice with 100 µL dig-wash buffer. We subsequently added 200 µL tagmentation buffer and incubated the samples at 37°C for 1 h. The reaction was stopped with 7 µL 0.5 M EDTA, 2 µL 10% SDS, and 1.7 µL 20 mg/mL proteinase K; for single-cell CUT&Tag assays, 5 µL stop buffer was used. CUT&Tag was performed using the Hyperactive In-Situ ChIP Library Prep Kit for Illumina (Vazyme Biotech). Embryos were incubated in 50 µL antibody buffer; 0.5 μg antibody was added and the mixture was incubated at 4℃ overnight. After washing twice with dig-wash buffer, 50 µL dig-wash buffer with 0.2 µg secondary antibody was added and the mixture was incubated at room temperature for 1 h. After washing twice with 100 µL dig-wash buffer, 1 µL pG-Tn5 and 49 µL dig-300 buffer were added, and the samples were incubated at room temperature for 1 h. Next, the samples were washed twice with 100 µL dig-wash buffer. We subsequently added 200 µL tagmentation buffer and incubated the samples at 37°C for 1 h. The reaction was stopped with 7 µL 0.5 M EDTA, 2 µL 10% SDS, and 1.7 µL 20 mg/mL proteinase K; for single-cell CUT&Tag assays, 5 µL stop buffer was used. After extraction with phenol-chloroform and ethanol precipitation, PCR was performed to amplify the libraries under the following conditions: 72°C for 3 min, 98°C for 30 s, 17 cycles of 98°C for 10 s and 60°C for 5 s, and a final extension at 72°C for 1 min with a hold at 4°C. For single-cell CUT&Tag of human embryos, CUT&Tag was performed in well of a microplate. After tagmentation, single human embryos were transferred to 200µl tubes containing 5% Proteinase K. After embryos lysis, PCR buffer and adaptors were directly added to tubes, normal PCR cycle was performed. Post-PCR clean-up was performed by adding a 1.5× volume of DNA Clean Beads (Vazyme Biotech).