Bioruptor 200 sonication system

MeDIP-qPCR was performed as previously described (6 (link)). Briefly, worm gDNA was fragmented to approximately 400 bp using a Bioruptor 200 sonication system (Diagenode). The fragmented DNA (15 μg) was immunoprecipitated with 5 μl of N⁶-mA antibody (Synaptic Systems, 202-003) at 4°C in a final volume of 500 μl containing MeDIP buffer [10 mM sodium phosphate (pH 7.0), 140 mM NaCl, and 0.05% Triton X-100]. Then, 40 μl of SureBeads starter kit protein G (Bio-Rad) was added and incubated at 4°C for 4 hours. Afterward, the beads were washed with MeDIP buffer (five times) and then digested with proteinase K digestion buffer [50 mM tris-HCl (pH 8.0), 10 mM EDTA, 0.5% SDS, and proteinase K (2.5 mg/ml)] for 3 hours at 50°C. The DNA was purified and used for qPCR. The sequences of the qPCR primers are listed in table S2. For each gene, triplicate biological samples and three technical replicates were conducted.

Worm gDNA was sonicated to 200 to 300 bp with a Bioruptor 200 sonication system (Diagenode). Then, adaptors were ligated to gDNA using a NEBNext Ultra II DNA library prep kit (Illumina). The ligated DNA was denatured at 95°C for 10 min. A portion of 10 μl of DNA was saved as input. Then, the gDNA was pulled down according to the MeDIP-qPCR protocol. After that, the N⁶-mA–enriched DNA fragments were purified. Then, immunoprecipitated DNA and input DNA were amplified by PCR with NEBNext Multiplex Oligos for Illumina following the manufacturer’s protocol and were then subjected to sequencing with Illumina HiSeq 4000 sequencing. MeDIP-seq was sequenced by Wuhan IGENEBOOK Biotechnology Co. Ltd. The adapter and low-quality reads were filtered out through Trimmomatic (version 0.38). Clean reads were mapped to the C. elegans genome by BWA (version 0.7.15), allowing up to two mismatches. Then, the data analysis was performed similarly to ChIP-seq using the same pipeline and software.