GSM2472869: OsNAR2.1RNAi MeDIP-seq; Oryza sativa; MeDIP-Seq
Genomic DNA were extracted from 10-day-old seedlings, and shipped to the Anoroad Genome company(Beijing) for whole-genome bisulfite sequencing(WGBS). WGBS librarie preparation, sequencing and data analysis was described as before. WGBS libraries are prepared for subsequent cluster generation starting from sample DNA through adaptor ligation, library purification, and quantification. Input gDNA (5 μg) is fragmented by sonication. The fragments are blunt-ended and phosphorylated, and a single ʹAʹ nucleotide is added to the 3ʹ ends of the fragments in preparation for ligation to a methylated adapter that has a single-base ʹTʹ overhang. The ligation products are purified and size-selected by agarose gel electrophoresis. Size-selected DNA is bisulfite-treated and purified. The treated DNA is PCR-amplified to enrich the fragment that have adapters on both ends. The final purified product is then quantitated prior to cluster generation. The WGBS libraries will be sequenced using Illumina HiSeq2500 /4000 Analyzer according to the manufacturer’s instructions. MeDIP sequecing in this study done by KangChen Bio-tech, Shanghai, China. Sequencing library preparation and data analysis was described as before (Ding et al 2015 ). Genomic DNA was sonicated to ~200 - 600bp with a Bioruptor sonicator (Diagenode). 800 ng of sonicated DNA was end-repaired, A-tailed, and ligated to single-end adapters following the standard Illumina genomic DNA protocol. After agarose size-selection to remove unligated adapters, the adaptor-ligated DNA was used for immunoprecipitation using a Rat monoclonal anti-5-methylcytosine antibody (Diagenode). For this, DNA was heat-denatured at 94°C for 10 min, rapidly cooled on ice, and immunoprecipitated with 1 μL primary antibody overnight at 4°C with rocking agitation in 400 μL immunoprecipitation buffer (0.5% BSA in PBS). To recover the immunoprecipitated DNA fragments, 100 μL of protein G magnetic beads (Life Technologies) were added and incubated for an additional 2 hours at 4°C with agitation. After immunoprecipitation, a total of five immunoprecipitation washes were performed with ice-cold immunoprecipitation buffer. A nonspecific Rat IgG immunoprecipitation was performed in parallel to methyl DNA immunoprecipitation as a negative control. Washed beads were resuspended in TE buffer with 0.25% SDS and 0.25 mg/mL proteinase K for 2 hours at 65°C and then allowed to cool down to room temperature. MeDIP and supernatant DNA were purified using Qiagen MinElute columns and eluted in 16 μL EB (Qiagen). 14 cycles of PCR were performed on 5 μL of the immunoprecipitated DNA using the single-end Illumina PCR primers. The resulting reactions were purified with Qiagen MinElute columns, after which a final size selection (300-700 bp) was performed by electrophoresis in 2% agarose. Libraries were quality controlled by Agilent 2100 Bioanalyzer. An aliquot of each library was diluted in EB to 5 ng/μL and 1 μL was used in real-time PCR reactions to confirm the enrichment for methylated region.
GEO Accession
GSM2472869