GSM9337271: BS-Nano - mESC treated with DNMTi rep3; Mus musculus; Bisulfite-Seq
Cells were resuspended in 300 μL cell lysis buffer (0.1 M Tris pH 8, 0.2 M NaCl, 5 mM EDTA, 0.1% SDS, and 10 units/mL Proteinase K) and incubated at 50 °C 1h to overnight. gDNA was precipitated with 600 μL of 100% isopropanol and incubated at -20 °C for 2 hours before centrifuging at 12,000 xg for 15 minutes. The supernatant was carefully discarded, and the remaining pellet was washed with 500 μL of 70% ethanol. Samples were again centrifuged at 12,000 xg for 15 minutes, supernatant was discarded, and the pellet was resuspended in 50 μL of water. Bisulfite treatment was performed using EpiTect Fast DNA Bisulfite kit (Qiagen 59824) following manufacturer's instruction. Bisulfite converted DNA was eluted in 15 μL of water. Target fragment was PCR amplified using EpiMark Hot Start Taq (NEB). 1.5 μL of eluted DNA was used for each reaction. PCR was performed at 95 °C 30 sec / 45X 95 °C 30 sec 45 °C 30 sec 68 °C 30 sec / 68 °C 5 min using bisulfite conversion-aware primers. PCR product was gel purified and pooled for sequencing at Plasmidsaurus using Premium PCR sequencing service.