GSM1213646: 71 (MBD-Seq); Rattus norvegicus; MBD-Seq
For tissue preparation, ether anesthetized animals were decapitated, the sculls opened and cortex removed for preparation of dorsal hippocampus. Five mm thick hippocampus slices were prepared along the septo-temporal axis and collected in ice cold PBS, thereby, removing blood contamination. Hippocampi were snap frozen in liquid nitrogen and stored at -80oC until further use. Each probe was individually processed and not pooled with other samples. For downstream applications, hippocampus was homogenized in ice cold 1x PBS and divided into equal volumes for DNA and RNA extraction. 10 mg rat hippocampal tissue was used for DNA extraction using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Briefly, 500 ng of rat hippocampal DNA from each animal was fragmented to a median size of 200-300 bp and subjected to methylated DNA capture according to the MethylMiner protocol (Invitrogen, Darmstadt, Germany). Enriched DNA was eluted at high salt concentrations (2 M NaCl). Sequencing libraries were prepared using the NEBNext® DNA Library Prep Reagent Set for Illumina® (New England Biolabs). Libraries were quantified on MultiNA system and sequenced at a concentration of 12 pM on the Genome Analyzer IIx (Illumina Inc, CA, USA) using the TruSeq version 5 cluster kit (cBot) and SBS kit (36 cycles).
GEO
GSM1213646