Truseq dna sample preparation procedures

Purified genomic DNA was sonicated to 200 base pairs (bp). 5 µg of fragmented genomic DNA was immunoprecipitated with 5 µg of rabbit 6mA antibody (Abcam) or rabbit IgG overnight at 4 °C in a final volume of 500 µl immunoprecipitation buffer (10 mM sodium phosphate, pH 7.0, 140 mM NaCl, 0.05% Triton X-100). The mixture was incubated with 50 µl protein A/G agarose beads for 2 h at 4 °C and then washed three times with 1 ml of immunoprecipitation buffer. The beads were then treated with proteinase K and the methylated DNA was purified by phenol–chloroform extraction followed by ethanol precipitation for library construction. End repair, 3′-adenylation, adaptor ligation, and PCR amplification were performed according to the Illumina TruSeq DNA sample preparation procedures. The libraries were sequenced using an Illumina HiSeq 2500 V4 platform. Of note, the anti-6mA used in this study can efficiently capture either single-strand or double-strand genomic DNA (Supplementary Figure 1b, c)
It is worth to note that both IgG- and input-based controls have been widely used to call peaks in DNA-IP-seq experiments in many studies^{31 (link),32} . In this study, we found that there was no significant bias between two approaches. For example, at the 0.75-h stage, we found that 96% of Input-controlled 6mA peaks were overlapped with IgG-controlled 6mA peaks (Supplementary Figure 1f).

Approximately 10 ng purified genomic DNA was digested in 0.5h or 12 h by using 20 U DpnI (NEB, Cat. no. R0176S). Digested DNA segments were further sheared by Bioruptor (Diagenode) to ∼300 bp. After purification by AMPure XP beads (Beckman Coulter, Cat. no. A63880), DNA segments were end-repaired, followed by 3′-adenylation and adaptor ligation according to standard Illumina TruSeq DNA sample preparation procedures. Adaptor-ligated DNA segments were PCR amplified for 15 cycles, purified by AMPure XP beads and suspended in 20 μl of resuspension buffer to yield the sequencing library. Quality control and concentration measurement were performed using Agilent 2100 Bioanalyzer DNA 1000 Chip and qPCR. Library was sequenced by using Illumina HiSeq 2500.

Plasmids were transfected into HEK293T cells using polyethyleneimine (PEI, Sigma). The cells were treated with lysis buffer (50 mM Tris-HCl, pH 7.4, 500 mM NaCl, 1% NP-40, protease inhibitor) after culturing for 48 h. Following centrifugation (20,000 × g) for 10 min at 4 °C, the supernatants were mixed with anti-FLAG M2 affinity beads (Sigma) and incubated for 3-h at 4 °C. Then the beads were washed twice with lysis buffer and twice with TBS (20 mM Tris-HCl, pH 7.4, 150 mM NaCl). Five micrograms of fragmented genomic DNA and immunoprecipitation buffer to a final volume of 500 µl was added to the beads and held overnight at 4 °C. Then the mixture was washed three times with 1 ml immunoprecipitation buffer and the beads were treated with proteinase K. The bound DNA was purified by phenol–chloroform extraction followed by ethanol precipitation. Sequencing libraries were prepared according to the Illumina TruSeq DNA sample preparation procedures and high-throughput sequencing was performed using an Illumina HiSeq 2500 V4 platform.