Bisulfite-seq of Cryphonectria parasitica: TdBCK1-S1
Input gDNA (5 Ľěg) is fragmented by Covaris shearing. The fragments are blunt?ended and phosphorylated, and a single ĄŽAĄŻ nucleotide is added to the 3ĄŻ ends of the fragments in preparation for ligation to a methylated adapter that has a single?base ĄŽTĄŻ overhang (using Truseq DNA library preparation kit, illumina). The ligation products are purified and size?selected by agarose gel electrophoresis. Size?selected DNA is bisulfite?treated and purified. The procedures were adapted from the EpiTect Bisulfite Kit (Qiagen). The treated DNA is PCR?amplified to enrich for fragments that have adapters on both ends. The final purified product is then quantified using qPCR according to the qPCR Quantification Protocol Guide and qualified using the Agilent Technologies 2100 Bioanalyzer.( Agilent technologies,Palo Alto CA, USA). And then we sequenced using the HiSeq˘â 2000 platform (Illumina,San Diego, USA)