GSM1541278: Sham 1 H3K27ac ChIP-seq; Rattus norvegicus; ChIP-Seq
At 72 h post-operation, rats were sacrificed and freshly dissected rat sciatic nerve was minced in 1% formaldehyde for 10min and then quenched for 10min with glycine to a final concentration of 0.125M. Samples were sequentially lysed in buffers LB1, LB2, and LB3 + 0.03% SDS (Schmidt et. al., 2009).   DNA was fragmented to an average size of 0.5-2kb using 4x10min Bioruptor (Diagenode) cycles on the medium setting.  Each aliquot of sonicated chromatin (300ug) was incubated overnight at 4°C with 5ug of antibody.  A 10% aliquot was saved for input analysis.  An 80ul aliquot of Protein G Dynabead (Invitrogen) slurry was added to each ChIP sample for 4hrs at 4°C.  IPs were washed 5x in RIPA buffer than eluted at 65°C with reverse crosslinking buffer (50mM Tris, 10mM EDTA, 1% SDS).    ChIP DNA was purified by phenol chloroform extraction and resuspended in 10mM Tris pH 8.0. Antibody used in the study: H3K27ac (Active Motif 37133) Libraries were prepared from sham/injured P25 sciatic nerve H3K27ac ChIP samples, and respective inputs, using the Illumina TruSeq ChIP Sample Preparation Kit.