title: GSM1547692: E16.5 Morc1 het H3K4me3 input; Mus musculus; ChIP-Seq
library_construction_protocol: The ChIP-seq protocol was adapted from published sources. FACS sorted cells from an individual mouse were diluted to 292μL with room temperature 1xPBS. 8.11μL 37% Formaldehyde (Sigma) was added and the sample was incubated 10 minutes at room temperature with rocking. 48.8μL of 1M glycine was then added to yield a final concentration of 0.14M and the samples were quenched 30 minutes with rocking. Cells were then spun 425g for 10 minutes at RT. The cell pellet was flash frozen. After thawing, the cells were resuspended in 200μL Lysis buffer (50mM Tris-Cl pH 8.0, 20mM EDTA pH 8.0, 1% SDS, 1x Complete Protease Inhibitor(Roche)) and incubated on ice ten minutes. Samples were then subjected to a nine minutes disruption using a Bioruptor on “High” setting, with 30s/30s off disruption (so 4.5 minutes of disruption total). Samples were spun 14000g 10 minutes to remove insoluble material. The soluble sample was diluted to 500μL with dilution buffer (16.7mM Tris pH 8, 0.01% SDS, 1.1% TritonX-100, 1.2mM EDTA, 167mM NaCl) and 10% of material was saved as input. Sample was precleared with 30μL Protein A Dynabeads (Life Technologies) and preincubated 1hr. The cleared material was incubated with 1μL L anti-H3K4me3 antibody (Millipore 04-745) overnight. The samples were incubated with 30μL Protein A Dynabeads and the precipitated material was recovered with a magnet. The beads were washed 2x4 minutes with Buffer A (50mM HEPES pH 7.9, 1% Triton X-100, 0.1% Deoxycholate, 1mM EDTA, 140mM NaCl), 2x4 minutes with Buffer B (50mM HEPES pH 7.9 0.1% SDS 1% Triton X-100 0.1% Deoxycholate, 1mM EDTA, 500mM NaCl) and 2x4 minutes with 10mM Tris/1mM EDTA. Bound material was eluted with 100μL Elution buffer (50mM Tris pH 8.0, 1mM EDTA, 1% SDS) at 65C for ten minutes and then eluted a second time with 150μL elution buffer. The input samples were thawed and diluted with 200μL buffer. Crosslinking of ChIP and input samples was reversed by incubating 16 hours at 65C. Samples were cooled and treated with 1.5μl of 10mg/mL RNaseA (PureLink RNAse A, Invitrogen #12091-021) for thirty minutes at 37C. 100μg of Proteinase K was then added and the samples treated for 2hr at 56C. The samples were then purified using a Qiagen Minelute kit. Samples were amplified by a SeqPlex DNA Amplification kit (Sigma) and then converted to libraries using an Ovation Rapid Library kit.