ChIP-seq for RelA of 10 ug/ml anti-IgM-stimulated mouse B cell: adult spleen
Splenic B cells in the presence or absence of anti-IgM (1 hour, 10 μg/ml) were used for ChIP-seq. ChIP was performed using the SimpleChIP Enzymatic Chromatin IP kit (Cell Signaling Technology, Beverly, MA) and experiments were carried out according to the manufacturer's protocol. In brief, 2 × 10^7 cells per samples are fixed with formaldehyde (Thermo Scientific, Waltham, MA). Chromatin is digested with Micrococcal Nuclease into 150-900 bp DNA/protein fragments at 37 degrees for 20 min, following this, five micrograms of Anti-NFkB p65 (Ab7970 abcam),and Normal Rabbit IgG (#2729 Cell Signaling Technology) was incubated with 50 μl of Dynabeads Protein G for 6 hr at 4 degrees. Cross-links are reversed, and DNA is purified with MinElute PCR Purification Kit (QIAGEN, Germany). The ChIP-seq libraries were prepared using TruSeq ChIP Library Preparation Kit (Illumina) following the manufacturer's protocol. 36 bp single end sequencing of the libraries was performed by HiSeq2500 (Illumina).