GSM4975536: SMC 5hmc 0hr replicate 1; Homo sapiens; Bisulfite-Seq
Cultured hcSMCs were washed once with PBS and lysed directly with the lysis buffer in the flasks, and total RNA and DNA were extracted using RNA/DNA/Protein Purification Plus Kit (Norgen #47700). The DNA samples was quantified using Qubit® 2.0 Fluorometer (Life technologies). One thousand ng of genomic DNA spiked with two pmol of biotinylated double-stranded oligonucleotides with 5-mC or 5-hmC modifications was treated with the 5mC Tet1 Oxidation Kit from Wise Gene according to the manufacture's instruction. After the reaction, DNA was purified using the QiaQuick PCR purification kit (Qiagen). The purified DNA was mixed with Dynabeads MyOne Streptavidin T1 and incubated at room temperature for 30 min with rocking. Both supernatant and beads were separately bisulfite-treated using EZ DNA Methylation-Gold Kit (Zymo Reesarch). The bisulfite-treated DNA from the beads was used for monitoring the conversion efficiency of 5-mC and 5-hmC. In contrast, the bisulfite-treated DNA from the supernatant was served for library preparation with the tPBAT protocol described in Miura F et al. (2019).