Methyl-DNA immunoprecipitation sequencing of 02428-2x (diploid rice) anthers, the anthers at the meiosis stage
We sonicated anthers DNA to produce random fragments ranging in size from 150 to 800 bp using the AIR™ DNA Fragmentation Kit (Bioo Scientific, Austin, Texas, USA). Based on the Illumina manufacturer’s recommended protocol, we then end-repaired, phosphorylated and A-tailed the fragmented DNA and ligated the adapters. Furthermore, the adaptor-ligated DNA was subsequent MeDIP enrichment using MethylMiner™ Methylated DNA Enrichment Kit (Life Technologies, New York, USA). The efficiency of enrichment was checked by real-time quantitative PCR. Then, PCR amplification was performed and excised target bands from the gel to produce libraries, and quantified these libraries by Agilent 2100 Analyzer. Finally, MeDIP-seq was performed on Illumina Hiseq2000 (LC Sciences, USA).