GSM4432411: THP-1_CTRL-input; Homo sapiens; OTHER
Genomic DNA was obtained from 5x10^6 cells with Cell/Tissue DNA Isolation Mini Kit (Vazyme) 50 ng genomic DNA extracted from THP-1 cell was fragmented into 300 bp reads by use of Ultrasonic Bioruptor sonicator (Diagenode Bioruptor). Then, the selective 5hmC chemical labeling was performed. The above fragmented DNA was added into 60 μl reaction buffer containing 6 μl of 10×β-GT reaction buffer, 2 μl of N3-UDP-Glc (3 mM), and 3 μl of β-GT (40 mM), and incubated at 37°C for 2 hr. The reaction product was purified with magnetic beads and DNA was eluted with 60 μl ddH2O. DMCO-S-S-PEG-Biotin (1 mM) was added into the DNA solution to reach a final concentration as 150 μM, and incubated at 37°C for 2 hr. The biotin labeled DNA was pulled down by 50 µl Dynabeads MyOne Streptavidin C1 beads (Life Technologies) for 30 min at room temperature. Next, the captured DNA fragments were subjected to 18 cycles of PCR amplification using Genomic DNA Sample Prep Kit (Illumina). The input library was prepared from fragmented DNA through direct PCR, without chemical labeling or capture. The libraries were denatured with 0.1 M NaOH to generate single-strand DNA (ssDNA) molecules and were added to the flow cell channel at a concentration of 8 pM. The libraries were quantified with a Qubit fluorometer (Life Technologies) and sequenced on a HiSEQ4000 sequencer (Illumina) with paired-end 50-bp reads.