GSM1568794: IgG MeDIP-Seq; Drosophila melanogaster; MeDIP-Seq
Tissue genomic DNA was extracted and purified with genomic DNA purification Kit (Promega) following the manufacturer’s instructions. To avoid RNA contaminations all samples were treated with RNase A two times. Purified genomic DNA was sonicated to 200 base pairs(bp). 5ug of fragmented genomic DNA was immunoprecipitated with 5 ug of rabbit m6A Antibody(millipore) or rabbit IgG overnight at 4°C in a final volume of 500uL of immunoprecipitation buffer (10 mM sodium phosphate at pH 7.0, 140mM NaCl, 0.05% Triton X-100). Then the mixture was incubated with 50uL of protein A/G Agarose beads for 2 h at 4°C and washed three times with 1 mL of immunoprecipitation buffer.The beads were then treated with proteinase K for 3 h at 50°C in 250μL proteinase K digestion buffer (50mM Tris–HCl, pH 8.0,10mM EDTA, 0.5% SDS), and the methylated DNA was purified by phenol-chloroform extraction followed by ethanol precipitation DNA libraries were prepared using the NEBNext® Fast DNA Library Prep Set For Ion Torrent(New England BioLabs) according to the instruction manual except that the input adaptor was reduced to 8µL and the adaptor ligated DNA was directly used in size selection without cleanup. Final libraries were purified with 12% native PAGE gel at 200 V for 40 min.