GSM5907275: Rel 0h_1 [ChIP-seq]; Mus musculus; ChIP-Seq
Cells were washed twice with PBS and then were fixed at room temperature with either 1% formaldehyde in PBS for 10 min (for H3K27ac and H3K4me3 ChIP) or 1.5mM EGS (Pierce Cat # 21565) for 30 min followed by 1% formaldehyde (Sigma-Aldrich) at room temperature for 15 min in PBS (RelA and Rel ChIP). Reactions were quenched by adding glycine to a final concentration of 0.125 M, and cells were washed twice with cold PBS. Nuclei were isolated and lysed in buffer containing 50 mM Hepes-KOH, pH7.5; 150 mM NaCl; 1mM EDTA; 1% Triton X-100; 0.1% sodium deoxycholate; 0.1% SDS; and protease inhibitors. The crosslinked chromatin was subjected to fragmentation by sonication (Branson Sonicator). ChIP was performed with 2.5 μg anti-RelA or anti-Rel antibody (Santa Cruz) and 3 μg H3K27ac and H3K4me3 antibody (Abcam) prebound to 50 μl Protein A or G Dynabeads (Invitrogen). Sonicated chromatin was added to antibody-bound beads and incubated at 4C overnight. Beads were collected by centrifugation, washed, and incubated at 65C for 4 h in elution buffer (50 mM Tris-HCl, pH 7.5; 10 mM EDTA; 1% SDS) to reverse cross-linking. ChIP DNA was purified by phenol-chloroform extraction followed by ethanol precipitation. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina HiSeq 2500 following the manufacturer's protocols.