title: GSM2824387: PFL_E11_WT_RAR; Mus musculus; ChIP-Seq
library_construction_protocol: Micro-dissected most proximal and distal tip domains of E11.5 forelimb buds from wild type CD1 mice were used for ChIP-seq. Samples were fixed in 1% formaldehyde/PBS for 20 minutes at room temperature, washed at least three times with cold PBS containing protease inhibitor and stored at -80°C. Pools of fifty pairs of wild type proximal or distal forelimb buds were used for each ChIP. 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. 20 µg of anti-RAR (sc-773X, Santa Cruz) and 3 µg of H3K27ac (ab4729, Abcam) 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.
attributes.tag: GEO Accession
attributes.value: GSM2824387