title: GSM5970648: control_Input_115; Mus musculus; ChIP-Seq
library_construction_protocol: H3K4me3 histone ChIP: For H3K4me3 based histone ChIP-seq analysis, craniofacial ectoderm was first isolated from E10.5 and E11.5 wild-type mouse embryos, as previously described (H. Li & Williams, 2013). Once isolated and pooled, tissue/chromatin was crosslinked with 1% formaldehyde at RT for 10 minutes. Following crosslinking, reactions were quenched using 0.125 M glycine, followed by multiple PBS washes.  Samples were subsequently frozen in liquid nitrogen and stored at -80°C. Once ~ 5 mg of tissue was collected per stage (e.g., E10.5, N = ~50 embryos; or E11.5, N = ~15 embryos), samples from multiple dissections, but similar stages, were pooled and combined with 300 µl of 'ChIP Nuclei Lysis buffer' (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS), with 1mM PMSF and 1X proteinase inhibitor cocktail (PICT, 100X from Thermo Scientific, Prod # 1862209).  Pooled tissue was resuspended completely and subsequently incubated at RT for 10 mins.  Following incubation, chromatin was fragmented using a Bioruptor (Diagenode, Cat. No. UCD-200) with the following settings: High energy, 30 seconds on, 30 seconds off, with sonication for 45 mins. Following shearing, chromatin was assessed as ~100-500 bp in size. Next, a small portion of fragmented chromatin was saved as input, while the rest was diluted 1 in 5 in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0, 5 mM EDTA, plus PMSF and PICT) followed by the addition of 20 µl protein A/G agarose beads (Pierce, Thermo Scientific, Prod # 20423 ) prewashed with RIPA buffer to eliminate non-specific binding. The pre-cleaned chromatin was then incubated with 2.5 µL of monoclonal H3K4Me3 primary antibody (Millipore, cat. #04-745), while rotating at 4°C, overnight. The following day, 20 ul protein A/G beads pre-saturated with 5 mg/ml BSA in PBS (Sigma, A-3311) were washed in RIPA buffer and subsequently added to the chromatin/antibody mix at 4°C, rotating, for 2 hours. Samples were then washed twice in RIPA, four times in Szak Wash (100 mM Tris HCl pH 8.5, 500 mM LiCl, 1% NP-40, 1% deoxycholate), twice more in RIPA followed by two TE washes (10 mM Tris HCl pH 8.0, 1 mM EDTA pH 8.0).  Finally, the bead slurry was resuspended in 100µl TE and the remaining bound chromatin was eluted off the beads using 200 ul 1.5X  'Elution Buffer' (70 mM Tris HCl pH 8.0, 1 mM EDTA, 1.5% SDS) at 65°C for 5 mins. Once eluted, crosslinks were reversed by incubating ChIP'd samples and input samples at 65°C overnight in 200 mM NaCl. Samples were then subjected to 20 ug of Proteinase K digestion at 45 ºC for 1h and DNA subsequently extracted using a standard Phenol:Chloroform, EtOH-precipitation based approach. Purified, pelleted, DNA was then resuspended in 20µl water. H3K4me3 histone ChIP-Seq library preparation and sequencing: Once purified fragments were obtained and quality and size confirmed, libraries were constructed using the Nugen ChIP Seq Library Construction Kit. Library purity, integrity, and size were then confirmed using High Sensitivity D1000 ScreenTape and subsequently sequenced using an Illumina MiSEQ platform and 50 bp single-end reads to a depth of ~25-30x106 reads per experimental sample and ~10x106 reads for input, carried out by the University of Colorado, Anschutz Medical Campus, Genomics and Microarray Core.