GSM6067811: WT testis D5-7, CUT&Tag, Chinmo, rep1; Drosophila melanogaster; OTHER
Cells were incubated with 10 μL pre-washed ConA beads in a 1.5-ml low-binding tube. Fifty microlitres of antibody buffer with 1 μg antibody (Rabbit anti-Chinmo-PA, 1:50) was added and cultured overnight at 4°C. After washing twice with dig-wash buffer, 50 μl dig-wash buffer with 0.5 μg secondary antibody was added and incubated at room temperature for 1 h. After washing twice with 800 μL dig-wash buffer, 1.0 μM pG–Tn5 was added with 100 μL dig-300 buffer. Samples were incubated at room temperature for 1 h and then washed twice with 800 μL dig-wash buffer. Three hundred microlitres of tagmentation buffer was added and samples were incubated at 37 °C for 1 h. The reaction was stopped with 10 μL 0.5 M EDTA, 3 μL 10% SDS and 2.5 μL 20 mg/ml Proteinase K. The NovoNGS ® CUT & Tag 2.0 High-Sensitivity Kit (Vazyme, Nanjing, China, N259-YH01) was used to capture Chinmo-binding sites. After extraction with phenol-chloroform and ethanol precipitation, PCR was performed to amplify the libraries according to the manufacturer's instructions. DNA quality and quantity were determined using the Agilent 2100 Bioanalyzer.