5hmC quantification

Quest 5-hmC DNA Elisa kit (Zymo Research, cat. nr. D5426) was used following the manufacturer’s instructions. Briefly, a 96 well plate was incubated with 1 ng/μl α-5-hydroxymethylcytosine antiserum in coating buffer for 1 hour at 37 °C. Wells were washed three times with 200 µl ELISA buffer, followed by blocking for 30 minutes at 37 °C with 200 µl of ELISA buffer. At the same time, genomic DNA (1 ng/μl) suspended in 100 µl ELISA buffer was heated to 98 °C for 5 min. Samples were immediately placed on the ice. DNA samples were than incubated in antibody-coated wells at 37 °C for 30 minutes. Wells were washed three times with ELISA buffer. α-ssDNA antiserum conjugated to HRP in ELISA Buffer (1:100 dilution) was incubated with each well for 30 min at 37 °C. Wells were washed three times with ELISA buffer and 100 µl of developer solution was added to each well; the color reaction proceeded for 30 min at 25 °C. Wells were analyzed for absorbance at 450 nm using a Wallac Victor² plate reader.