GSM3418004: Prdm16 KO  H3K27ac ChipSeq; Mus musculus; ChIP-Seq
Wt and KO Crypts were isolated from 3 mice each, pooled, and fixed with 1.l% formaldehyde for 15 min at room temperature and then quenched  with 2.5 M glycine for 5 mins at room temperature. Samples were washed with PBS, pelleted and resuspended in ChIP lysis buffer (0.6% SDS, 1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 20 mM Tris at pH 8) with protease inhibitors (Roche) and 1 mM PMSF. Crypts were sonicated to shear chromatin using an Active Motif EpiShear platform (8 minutes active time, 30 sec on, 20 sec off, amplitude 25%). To a achieve a final concentration of 0.1% SDS, chromatin was diluted 1:6 with ChIP dilution buffer (1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 20 mM Tris at pH 8).  Inputs were removed.  Chromatin was split into  primary antibodies were added for overnight incubation at 4°C with rotation with 3ug anti H3K27Ac (Active Motif, 39133). Protein A Sepharose beads (GE healthcare) were then added for 4 hours at 4°C. Samples were washed twice with wash buffer 1 (0.1% SDS, 0.1% NaDOC, 1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 20 mM Tris at pH 8), once with wash buffer 2 (0.1% SDS, 0.1% NaDOC, 1% Triton X-100, 0.5 M NaCl, 1 mM EDTA, 20 mM Tris at pH 8), once with wash buffer 3 (0.25 M LiCl, 0.5% NaDOC, 0.5% NP-40, 1 mM EDTA, 20 mM Tris at pH 8), and twice with wash buffer 4 (1 mM EDTA, 20 mM Tris at pH 8).  Samples were then eluted from beads with warm 100 mM NaHCO3/1% SDS buffer. Samples were reverse cross-linked overnight at 65°C with RNase A and then treated with proteinase K and column purified (Clontech NucleoSpin). ChIP enrichment was calculated as percent of input. H3K27 ChIP WT and KO samples were prepared as ChIPseq libraries and sequenced using a HiSeq 2000 high output sequencer.