Supplementary Figure 8: AI labeling of 5fC and subsequent C-to-T transition.
From: Bisulfite-free, base-resolution analysis of 5-formylcytosine at the genome scale
(a) Biotin conjugation for pulldown and DTT cleavage. (b) Sanger sequencing results for the fC-CET treated 5fC-containing 76-mer DNA, introducing the C-to-T transition during PCR amplification. (c) FspI-digestion tests to confirm the C-to-T transition caused by AI-mediated 5fC labeling. PCR-amplified input 70-mer 5fC oligo with FspI restriction site in the middle can be cleaved, whereas amplified products of 5fC-AI-Biotin or 5fC-AI-SH stay intact, suggesting loss of restriction site and hence C-to-T transition. (d,e) Efficiency of 5fC labeling on a model DNA. T%, calculated from TOPO-cloning (d) or high-throughput sequencing using Mi-Seq (e), was used to measure the C-to-T transition rate of 5fC after AI labeling and AI-mediated pulldown. In both cases, a normal C next to the target 5fCpG was used as a control.
