Analysis of DNA methylation

DNA from crude cell lysates was bisulfite converted and purified using EZ DNA Methylation-Gold Kit (Zymo Research Europe, Freiburg, Germany) according to the manufacturer's protocol. Genomic fragments encompassing sgRNA target sites were amplified from bisulfite converted DNA using the PyroMark PCR kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Methylation of repetitive LINE-1 elements was analyzed as described previously (37). The cycling was performed as follows: initial denaturation for 15 min at 95°C; 50 cycles of 30 s at 95°C, 30 s at 48°C (BACH2–A and BACH2–B fragments), 52°C (IL6ST–A fragment) or 64°C (LINE-1) and 30 s at 72°C; final extension for 10 min at 72°C. In order to quantify methylation levels at individual consecutive CpG sites, PCR amplicons were sequenced using the PyroMark Q24 Advanced pyrosequencing system (Qiagen). Sequences of PCR primers and pyrosequencing primers are listed in Supplementary Table S2.