GSM7697732: Mock_N_hmC_4; Mus musculus; OTHER
DNA was extracted by adding equal volume of buffered phenol:chloroform:isoamyl alcohol (25:24:1 ratio) and centrifuged in Phase-Lock tubes at 15,000 RPM at room temperature. Supernatant was transferred to clean tubes. An equal volume of isopropanol was then added to the supernatant and mixed well at room temperature to precipitate the DNA. The DNA was then centrifuged at 10,000g for 10 minutes at room temperature, and then washed in 70% ethanol. After all ethanol was removed, the DNA pellet was eluted in nuclease-free water and incubated at 55 for 1 hour before storing at -20. 5mg of genomic DNA was sonicated to 300-400 base pairs and 5hmC containing fragments were glucosylated (T4 phage ß-glucosyltransferase enzyme and UDP-6-N3-glucose). The glucosylated fragments were purified and then biotinylated (disulfide biotin linker) and pulled down with Dynabeads MyOne Streptavidin C1 beads. The 5hmC fragments were released from the beads using dithiothreitol and purified for a final time. DNA fragments were eluted in nuclease-free water and quantified by Qubit.