DNA extraction, bisulfite conversion, and pyrosequencing

Genomic DNA was extracted from different gRNA transgenic cell lines using DNA lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM EDTA pH 8.0, 100 mM NaCl, 1 % SDS) supplemented with Proteinase K (20 mg/ml). Briefly, the cell pellet was incubated with 500 µl DNA lysis buffer and 12.5 µl Proteinase K at 55°C overnight. The next day, 210 µl saturated NaCl solution (5 M) was added and centrifuged for 30 min. Then, the supernatant was transferred into a new tube, mixed with 700 µl isopropanol, and incubated for 10 min at room temperature. Samples were then centrifuged for another 30 min, the supernatant was discarded and the pellet was washed with 70% ethanol followed by another centrifugation step for 30 min. After this step, the supernatant was discarded and the dried DNA pellet was dissolved in TE buffer (1 mM EDTA pH 8.0, 10 mM Tris-HCl pH 8.0) and stored at 4°C. All centrifugation steps were performed at 14000 rpm at room temperature.

Bisulfite-treated DNA samples were prepared using DNA Methylation-Gold kit (Zymo Research, Irvine, CA, US) according to the manufacturer's protocol. Specific regions of the MGMT promoter were amplified with biotinylated primers using PyroMark PCR kit (Qiagen, Düsseldorf, Germany). Pyrosequencing was performed using PyroMark Gold Q96 Reagents on the PyroMark Q24 Sequencer System (Qiagen, Düsseldorf, Germany) according to the manufacturer's recommendation. The data of pyrosequencing were analyzed by PyroMark Q24 Software (Qiagen, Düsseldorf, Germany). The sequence of primers, conditions for PCR amplification, and pyrosequencing assays are shown in Supplementary Table S3.