title: GSM1833830: LMP2a-H3K27ac; Mus musculus; ChIP-Seq
library_construction_protocol: Mouse splenic B220+ B cells were cross-linked in 1% (vol/vol) formaldehyde, lysed in Lysis Buffer [50 mM Tris•HCl, pH 8.1, 10 mM EDTA, 1% SDS, and protease inhibitors (Roche)] and sonicated for 20 × 20-s cycles on ice. Lysate was diluted 10-fold with Dilution Buffer (16.7 mM Tris•HCl, pH 8.1, 1.2 mM EDTA, 167 mM NaCl, 1.1% Triton X-100, 0.01% SDS, and protease inhibitors) and ChIP grade rabbit polyclonal antibodies (Abcam) against Histone H3 (mono methyl K4), Histone H3 (tri methyl K4) and Histone H3 (acetyl K27) were added to immune precipitate H3K4me1-DNA, H3K4me3-DNA and H3K27ac-DNA complexes, respectively. Protein-DNA complexes were captured with the addition of protein A agarose/salmon sperm DNA and then washed one time for 15 min at 4 °C with rotation, with Low Salt Wash Buffer (20 mM Tris•HCl, pH 8.1, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1% SDS), one time with High-Salt Wash Buffer (20 mM Tris•HCl, pH 8.1, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 1% SDS), one time with LiCl Wash Buffer (10 mM Tris•HCl, pH 8.1, 1 mM EDTA, 0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate acid), and two times with TE buffer. Protein–DNA complexes were eluted from the protein A-beads by rotating at room temperature two times for 15 min in 250 μL of Elution Buffer (100 mM NaHCO3, 1% SDS) to a final volume of 500 μL. DNA–protein cross-link was reversed by the addition of 20 μL of 5 M NaCl and incubation at 65 °C for 4 h. DNA was purified using a PCR MinElute column (Qiagen). Libraries were prepared using ChIP-Seq Library Prep Reagent Set for Illumina from NEB. Briefly, DNA was end-repaired. 3' A base was added and ligated to adaptors. After PCR amplification, DNA was purified and analyzed with Illumina HiSeq.