P-GSE24178-6 - P-GSE24178-6
nucleic acid library construction protocol
Cells were fixed with 1% paraformaldehyde at 37°C for 10 minutes followed by quenching with 0.125 M glycine (final concentration). Chromatin was sheared by sonication to a size of 200-500bp which was then precleared with 40 μl protein A or protein G magnetic beads, followed by incubation overnight at 4 °C in RIPA buffer (10 mM Tris, pH 7.6, 1 mM EDTA, 0.1% (wt/vol) SDS, 0.1% (wt/vol) sodium deoxycholate and 1% (vol/vol) Triton X-100). Beads were washed twice with RIPA buffer alone, then twice with RIPA buffer plus 0.3 M NaCl, twice with LiCl buffer (0.25 M LiCl, 0.5% (vol/vol) Igepal CA630 and 0.5% (wt/vol) sodium deoxycholate), once with Tris-EDTA buffer (Tris 10 mM, pH 8.0, and 1 mM EDTA) plus 0.2% (vol/vol) Triton X-100, and once with Tris-EDTA buffer alone. ChIP DNA was extracted for 4 h at 65 °C in Tris-EDTA buffer with 0.3% (wt/vol) SDS and proteinase K (1 mg/ml) before processing with standard Illumina library preparation protocols.