GSM2572152: WCE CKp25 2wk rep1; Mus musculus; ChIP-Seq
Mouse hippocampus was harvested immediately after euthanasia. Upon dissection tissue was flash-frozen in liquid nitrogen. The tissue was minced and crosslinked with 1% formaldehyde at room temperature for 15mins and quenched with 0.125M Glycine. The tissue was then homogenized in cell lysis buffer using the Tissue Lyser and a 5mm stainless steel bead. Then the nuclei were lysed in cell lysis buffer and chromatin was sheared by sonication. Chromatin was incubated overnight at 4C with the H3K9Ac antibody. Immune complexes were captured with Protein A-sepharose beads, washed and eluted. Enriched chromatin was then subjected to crosslink reversal and proteinase K digestion at 65C, phenol-chloroform extraction and ethanol precipitation. Sequencing libraries were prepared from ~1-5 ng ChIP (or input) DNA. Gel electrophoresis was used to retain library fragments between 300 and 600 bp. Prior to sequencing, libraries were quantified using Qubit (Invitrogen) and quality-controlled using Agilent’s Bioanalyzer. Single end sequencing was performed using the Illumina HiSeq 2000 platform according to standard operating procedures.