Oligonucleotides containing 5fC, 5mC, 5hmC or 5caC were synthesized using the ABI Expedite 8909 Nucleic Acid Synthesizer. The modified nucleotides were site-specifically incorporated at desired positions (Supplementary Table 1) with commercially available phosphoramidites (Glen Research). Subsequent deprotection and purification were carried out with Glen-Pak Cartridges (Glen Research) following the manufacturer’s instructions. Purified oligonucleotides were characterized by MALDI-TOF (< 40-mer). Regular oligonucleotides (and PCR primers) were purchased from Sangon Biotech.
Long duplex DNAs (Supplementary Table 1 and 2) were prepared through ligation of short duplexes (20–40 bp) with sticky overhangs21 (link). In brief, the ligation-site oligonucleotides were phosphorylated with T4 polynucleotide kinase (NEB) and then annealed with the corresponding complementary strands. Annealed duplexes with sticky overhangs were mixed and ligated with T7 DNA ligase (NEB) at 16 °C for 4 h, followed by purification with native PAGE (10%).
“10% 5fC” dsDNA and “5xC mix” dsDNA used in the qPCR assay were prepared through PCR amplification as previously described18 (link). All modified dCTPs were purchased from Trilink.