title: GSM3414996: Sox4_ChIP-seq_1; Mus musculus; ChIP-Seq
library_construction_protocol: Nuclei were prepared from approximately 100 million cells and lysed in the sonication buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, and 1% SDS). Chromatin was sheered by sonication in iced water (Bioruptur XL, Diagenode), 30 sec on/30 sec off, for 35 min, to achieve 100–500-bp DNA fragments. After dialysis against the low SDS buffer (20 mM Tris-HCl pH 8, 10 mM EDTA, and 0.01% SDS) overnight, the sonicated samples were centrifuged at 20,000 g for 10 min. The soluble whole-cell extracts were added with Triton X-100 (final 1%) and NaCl (final 150 mM), and incubated with 100 µl of Dynal Protein G magnetic beads that had been pre-incubated with 10-µg mouse anti-FLAG antibody (Sigma) or mouse IgG. Beads were washed 1´ with low-salt immune complex wash buffer (0.1% SDS, 1% TritonX-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH8.1), 1´ with high-salt immune complex wash buffer(0.1% SDS, 1% TritonX-100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris-HCl, pH8.1), 1´ with LiCl immune complex wash buffer (250 mM LiCl, 0.5% NP-40, 0.5% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1), and 1´ with TE containing 50 mM NaCl. Bound complexes were eluted from the beads by heating at 65oC for 30 min (50 mM Tris-Hcl, pH 8.0, 10 mM EDTA, and 1% SDS) in a Thermomix. IgG ChIPs were performed under the same conditions as the experimental ChIPs. Crosslinking was reversed by incubating samples at 65oC for 6 hr. Whole-cell extract DNA reserved from the sonication step was treated in parallel to reverse crosslinking. After crosslink reversal, samples were treated with RNase and proteinase K prior to DNA extraction. Rubicon ThruPlex DNA-Seq library preparation system
attributes.tag: GEO Accession
attributes.value: GSM3414996