GSM2044667: 5hmC-Control 3; Mus musculus; MeDIP-Seq
Whole brains were extracted and immediately flash frozen in 2-methylbutane and dry ice. Whole hippocampal tissue was excised by micropunch (− 0.95 to − 3.79 mm posterior to bregma) and approximately 30 mg of tissue was homogenized with glass beads (Sigma) and DNA and RNA were extracted using AllPrep DNA/RNA mini kit (Qiagen). Chemical labeling-based 5hmC enrichment was described previously ( Song et al., 2011 and Papale et al., 2015). Briefly, a total of 10 μg of pooled hippocampal DNA (N = 2 mice for all enrichments, except N = 1 for one of the enrichment) was sonicated to 300 bp and incubated for 1 h at 37 °C in the following labeling reaction: 1.5 μl of N3-UDPG (2 mM); 1.5 μl β of -GT (60 μM); and 3 μl of 10 × β-GT buffer, in a total of 30 μl. Biotin was added and the reaction was incubated at 37 °C for 2 h prior to capture on streptavidin-coupled Dynabeads (Invitrogen, 65001). Enriched DNA was released from the beads during a 2-hour incubation at room temperature with 100 mM DTT (Invitrogen, 15508013), which was removed using a Bio-Rad column (Bio-Rad, 732–6227). Capture efficiency was approximately 5–7% for each sample. 5hmC-enriched libraries were generated using the NEBNext ChIP-Seq Library Prep Reagent Set for Illumina sequencing, according to the manufacturer's protocol. Briefly,5hmC-enriched DNA fragments were purified after the adapter ligation step using AMPure XP beads (Agencourt A63880). An Agilent 2100 BioAnalyzer was used to quantify the amplified library DNA and 20 pM of diluted libraries (N = 6) were used in each lane for sequencing, which yielded approximately 25 to 35 million uniquely mapped 50 bp reads from each library. 50-Cycle single-end sequencing was performed by Beckman Coulter Genomics. Image processing and sequence extraction were done using the standard Illumina Pipeline.
GEO Accession
GSM2044667