title: GSM3386343: ChIP_H3K4me1_CPP_2; Mus musculus; ChIP-Seq
library_construction_protocol: To prepare chromatin extract, micro-dissected tissue from E9.5 CD-1 mouse embryos (2x from 50 embryo pools each) obtained from Charles River were cross-linked in PBS containing 1% formaldehyde at 25°C for 10 minutes. The reaction was quenched by 125 mM glycine. The cross-linked tissues were incubated in LB1 (50 mM HEPES-KOH, pH 7.5; 140 mM NaCl; 1 mM EDTA; 10% Glycerol; 0.5% NP-40; 0.25% Triton X-100) with protease and phosphatase inhibitors on ice for 10 minutes. The tissues were then sonicated in LB3 (10 mM Tris-HCl, pH 8.0; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% sodium deoxycholate; 0.5% N-lauroyl sarcosine; 1% Triton X-100) with protease and phosphatase inhibitors. Chromatin extract was then cleared by centrifugation at 14,000g, 4°C for 10 minutes. For immunoprecipitation, the chromatin extract was incubated with 5ug of the anti-TBX5 antibody (Santa Cruz Biotechnology sc-17866; Lot #G1516), 1μg of anti-H3K4me3 (Wako Chemicals #305-34819; Lot #14004), or 1μg of anti-H3K4me1 (Abcam ab8895; Lot #GR257926-1) at 4°C for >12 hours in a total volume of 200 μL. The immune-complexes were captured by Protein G-conjugated magnetic beads (Life Technologies, 1003D), washed in sequence by LB3, LB3 with 1 M NaCl, LB3 with 0.5 M NaCl, LB3, and TE (10 mM Tris-HCl, pH 8.0; 1 mM EDTA). The captured chromatin was eluted in ChIP Elution Buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA; 1% SDS; 250 mM NaCl) at 65°C. After RNase and proteinase K treatment and reverse cross-linking, DNA were purified. High-throughput sequencing libraries from ChIP and input DNA were prepared using NEBNext Ultra DNA Library Prep Kit (New England Biolabs, E7370S). During library preparation, adaptor-ligated DNA fragments of 200-650 bp in size were selected before PCR amplification using Sera-Mag magnetic beads (GE, 6515-2105-050-250). DNA libraries were sequenced using Illumina Hi-seq instruments (single-end 50 base) by the Genomics Core Facility at the University of Chicago.
attributes.tag: GEO Accession
attributes.value: GSM3386343