Instrument: Illumina Genome Analyzer IIx
Strategy: MBD-Seq
Source: GENOMIC
Selection: MBD2 protein methyl-CpG binding domain
Layout: SINGLE
Construction protocol: Tissue was collected at 3 months after induction of status epilepticus. For tissue collection rats were anesthetized with CO2 and decapitated with guillotine. Left hippocampus was rapidly isolated and tissue containing CA3 and dentate gyrus was homogenized in ice cold 1x PBS and divided into equal volumes for DNA and RNA extraction. Samples were stored at -80oC until use. 2000 ng of hippocampal DNA from each animal was fragmented to median size of 200-300 bp and subjected to methylated DNA capture according to MethylMiner protocol (Invitrogen, Darmstadt, Germany), exclusively enabling capture of methylated double stranded DNA. Fragmented and enriched DNA was eluted at high salt concentrations (2M NaCl). 5ng of enriched DNA was used in library preparation using the NEBNext DNA Library Prep Reagent Set for Illumina (New England Biolabs, Frankfurt/Main, Germany). Quality of sequencing libraries was assayed using the Shimadzu MultiNA capilary electrophoresis system (Shimadzu, Kyoto, Japan). Libraries were sequenced at a concentration of 10 pM on the Illumina Genome Analyzer IIx (Illumina, San Diego, CA, USA) with a 36 bp single read length.