title: GSM6723061: Sperm_01; Mus musculus; OTHER
library_construction_protocol: Single cells were isolated into lysis buffer and then heat for 30min at 75℃. After that, the DNA was treated with protease. Cells were lysed and treated by protease. Then naked DNA was tagmented by indexed Tn5 at 55 °C for 15 min (Vazyme Biotech).Pool 8 cell lysate into one tube, denature transposons at 72℃. T4 DNA ligase buffer and 1μL Exo I were added and lysate incubate at 37℃ for 60 min.DNA were purified with the 2× AMPure (Beckman) beads with 500ng carrier RNA (thermo).dC-tailing was performed according to previous protocol.Potassium perruthenate (Sigma-Aldrich,334537) solution was prepared according to published protocol.To obtain the 5hmC profiles from the single cells, the 5hmC labeled DNA was subjected to multiple rounds of primer extension to record the 5hmC-to-T transition signal.Primer extension mix (8 µL of 5× KAPA 2G buffer, 0.8 µL of 10 mM dNTPs, 0.8 µL of 10 µM P5G9H primer, 0.6 µL KAPA2G Robust HS DNA polymerase (Roche, KK5023)) was added.Add 8 µL of KAPA HiFi buffer, 2 µL of 500nM P5-short, 2 µL of 500nM P7-short 1.6 µL Kapa HF Hotstart DNA polymerase directly to the linear amplification mixture. PCR was performed to amplify the library for 11-15 cycles.After the PCR reaction, libraries were purified with the 0.8× AMPure beads. schmC-CATCH