COBRA and bisulfite sequencing

Genomic DNA (100 ng) was bisulfite-converted with the EpiTect bisulfite kit (Qiagen). The target regions were amplified by touchdown PCR with the Platinum Taq DNA Polymerase (Thermo Fisher Scientific) using the following conditions: 20 cycles of 30 s at 95 °C, 30 s at 60–50 °C (with a 0.5 °C decrease per cycle), 50 s at 72 °C followed by 35 cycles of 30 s at 95 °C, 30 s at 50 °C, and 50 s at 72 °C. The PCR products were purified using the PCR cleanup kit (Macherey Nagel). For COBRA, 40 ng of PCR product were digested by Taq1α (Thermo Fisher Scientific) and loaded on an agarose gel alongside 40 ng of undigested PCR product. For bisulfite sequencing, the PCR products were cloned by TA cloning in the pCR2.1 vector (Thermo Fisher Scientific) and sequenced by Sanger sequencing (Eurofins). Sequences were aligned with the BISMA software and filtered to remove identical clones. The primers are provided in the Supplementary Data ⁷.