MeDIP of  resistant NIL HD2329 +Lr28 at 96hai
(i) 1 g of the genomic DNA was sheared independently for each of the four treatments and end repaired to obtain sheared fragments with blunt ends. (ii) A single A nucleotide was then added to the 3 ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction. A corresponding single T nucleotide on the 3 end of the adapter provides a complementary overhang for ligating the adapter to the fragment. This strategy ensures a low rate of chimera (concatenated template) formation. (iii) The multiple indexing adapters were then ligated to the ends of the DNA fragments to prepare them for hybridization on to the flow cell. (iv) This was followed by immunoprecipitation using MeDIP buffer and 5mC specific monoclonal antibody. This immunoprecipitated DNA is supposed to be enriched for methylated DNA fragments, which had adapter molecules on both ends for amplification of the DNA in the library. (v) The PCR was performed with the PCR Primer Cocktail that annealed to the ends of the adapters. The number of PCR cycles was kept to the minimum to retain library representation. (vi) Qubit concentration of library was checked followed by quality check of the library; this was done by loading 1l of sample on an Agilent Technologies 2100 Bioanalyzer using a DNA-specific chip (e.g., Agilent DNA 1000 or DNA HS chip). (vii) The MeDIP library obtained thus was sequenced and the data was prepared in the form of FASTA files and reads quality checked using the following parameters: Base/sequence quality score: Phred score >30; average base content per read: 100bp; GC distribution:>40% and over-represented sequences. Based on the quality of sequence reads, trimming was done, wherever necessary, to retain only high quality sequence.