GSM7332679: 21d Nkapl KO1 DRIP input; Mus musculus; OTHER
Nuclei was isolated from testis tissues before SDS/proteinase K treatment at 37°C for 4-6 hours, followed by genomic DNA extraction with the phenol:chloroform:isoamyl alcohol method. The precipitated DNA pellet was washed, air dried, resuspended in TE buffer, and quantified by the Qubit dsDNA HS Assay kit (Invitrogen, Q32854). 10 μg gDNA was digested with a combination of restriction enzymes of MseI, DdeI, AluI, and MboI at 37°C overnight, and the negative control was treated with RNase H (New England Biolabs, M0297S). For DNA:RNA hybrids immunoprecipitation (DRIP), 5 μg gDNA was resuspended in 500 μl TE buffer. 450 μl gDNA mixture was incubated with 10 µg S9.6 antibody for DNA:RNA immunoprecipitation, and the remaining 50 μl was used as input DNA. Protein G dynabeads were pre-washed for three times with 1×DRIP binding buffer (100 mM sodium phosphate, pH 7.0; 1.4 M NaCl; 0.5% Triton X-100) and then added to the DNA-antibody mixtures with gentle shaking at 4 °C for 3~4 hours. DNA was eluted with DRIP elution buffer, and then sheared to 250 bp in size by sonication using the Covaris-S220. These DNA samples was denatured at 95°C for 2 mins and then incubated on ice for 2 mins to obtain ssDNA fragments. ssDRIP-seq libraries were constructed by using the VAHTS ssDNA Library Prep Kit for Illumina (Vazyme, ND620-02), following instructions from the manufacturer.