GSM8860444: Ctrl-Sealseq; Homo sapiens; OTHER
Genomic DNA was obtained from 5x106 cells with Cell/Tissue DNA Isolation Mini Kit (Vazyme) 50 ng genomic DNA were fragmented in Tagmentation buffer at 55 °C. Fragmented DNA was purified with Zymo DNA Clean and Concentration Kit (Zymo). Then, the selective 5hmC chemical labeling was performed. The above fragmented DNA was added to glucosylation buffer (50 mM HEPES buffer, 25 mM MgCl2, pH 8.0) containing β-GT and N3-UDP-Glc, and incubated at 37°C for 2 hours. T After DNA purification, DBCO-PEG4-Biotin (Click Chemistry Tools) was added into the DNA and incubated at 37°C for 2 hours. The biotin-labeled DNA was pulled down by C1 Streptavidin beads (Life Technologies) for 15 min at room temperature. Next, the captured DNA fragments were subjected to PCR amplification using Nextera DNA sample preparation kit (Illumina). The amplified PCR products were purified by 1.0x AMPure XP beads (Acegen). The input library was prepared from fragmented DNA through direct PCR, without chemical labeling or capture. The libraries were quantified with a Qubit fluorometer (Life Technologies) and sequenced on Illumina Novaseq with 150-bp paired-end reads.