GSM2061133: DFL_E12_WT_Input_rep1; Mus musculus; ChIP-Seq
Micro-dissected proximal and distal segments of E12.5 limbs either from wild type CD1 mice, or from various combinations of Hoxa13;Hoxd13 mutant alleles and of HH28 forelimbs from chick were used for ChIP-seq. Samples were fixed in 1% formaldehyde/PBS for 18 minutes at room temperature, washed at least three times with cold PBS containing protease inhibitor and stored at -80°C. Pools of ten pairs of wild type proximal or distal forelimbs, or of three pairs of Hoxa13-/-;Hoxd13-/- double mutant proximal or distal fore- and hindlimbs, or thirteen pairs of chick proximal or distal forelimbs were used for each experiment. The samples were treated with lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl and protease inhibitor) for 10 min on ice, followed by RIPA buffer (TE with 140 mM NaCl, 0.1% deoxycholate, 0.1% SDS, 1% TritonX-100 and protease inhibitor) and fragmented to a range of 200-500 bp by a Biorupter sonication device (Diagenode). PierceTM Protein A/G magnetic beads (88803, Thermo Scientific) were conjugated with each antibody in blocking buffer (PBS, 0.5% Tween20, 0.5% BSA and protease inhibitor) for 4 hrs at 4°C and washed with blocking buffer. Chromatin was incubated with the prepared protein A/G beads overnight at 4°C with rotation. 4 µg of anti-HOXA13 (PMID: 7590231), 3 µg of H3K27ac (ab4729, Abcam) and 3 µg of H3K27me3 (07-449, Millipore) were used for each immunoprecipitation. Samples were washed 6 times with RIPA buffer, twice with RIPA/500 mM NaCl buffer, twice with LiCl wash buffer (TE, 250 mM LiCl, 0.5% NP-40 and 0.5% deoxycholate) and twice with TE, followed by eluted in direct elution buffer (10 mM Tris-HCl, 5 mM EDTA, 300 mM NaCl and 0.1% SDS). Cross-links were reversed overnight at 65°C and ChIPed DNA was treated with RNase A and proteinase K, and purified by phenol chloroform extraction. For ChIP-seq, at least 5 ng of purified DNA were used to make libraries according to the manufacturer’s protocol (Illumina). The material was sequenced with 100 bp single-end reads on the Illumine HiSeq according to the manufacturer’s specifications.
GEO
GSM2061133
GEO Accession
GSM2061133