GSM8493504: HDI ChIP; Mus musculus; ChIP-Seq
Cells were homoginised and cross-linked in 1% formaldehyde for 20 min at room temperature. Whole-cell extracts were sonicated followed by immunoprecipitation with antibodies for H3k27ac (Abcam, ab4729) and Protein A Sepharose CL-4B (GE, 17-0780-01). After the immunoprecipitation, beads were washed by ChIP dilution buffer (50 mM HEPES (pH 7.5]), 155 mM NaCl, 1.1% TritonX-100, 0.11% NaDeoxycholate, 1 mM EDTA) three times followed by a wash with ChIP wash buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.5% NaDeoxycholate, 250 mM LiCl, and 0.5% NP-40). Sepharose beads were further washed once by TE buffer (Promega, E260A). DNA were enriched with phenol–chloroform extraction and dissolved in 20 µl TE buffer followed by RNA degradation by RNase (Roche, 11119915001) for 30 min at 37 °C. Four HDAC3 ChIP reactions were performed with each reaction containing samples from three mice. The ChIP reactions were then pooled to be sequenced as one sample, together with the pooled total input DNA as the control. The precipitated DNA was then pooled and amplified according to the manufacturer's guide of Illumina (Ilumina TruSeq ChIP Library Preparation Kit), followed by 50 bp single-end deep sequencing on Illumina NovaSeq 6000.