title: GSM2458154: Littermate control 8h LPS RelA ChIP-seq; Mus musculus; ChIP-Seq
library_construction_protocol: For the RelA and Ikaros ChIP, BMDMs were cross-linked with 1% formaldehyde for 10 min at room temperature. After cross-linking, chromatin was isolated from approximately 2 × 107 cells and processed as follows. The lysis buffer to shear the chromatin contained 0.5% SDS, 10 mM EDTA (pH 8), 50 mM tris-HCl (pH 8), and proteinase inhibitor cocktail. Sonication was performed with a Bioruptor Plus (Diagenode) at medium setting for a total time of 17 min for Ikaros ChIP, 20 min for RelA ChIP, using a 30 sec ON, 30 sec OFF cycle to shear the chromatin to generate DNA fragments with a size range of 200 to 600 base pairs. The sonicated mixture was centrifuged at 14,000 rpm for 10 min. The sheared chromatin samples were diluted 1:5 in dilution buffer [0.01% SDS, 1.1% Triton-X, 1.2 mM EDTA (pH 8), 20 mM tris-HCl (pH 8), 167 mM NaCl, and proteinase inhibitor cocktail]. For the RelA ChIP, 10 ml of a rabbit polyclonal anti-RelA antibody (ab7970, Abcam) was diluted in 1 ml immune complex buffer with Dynabeads protein A (Invitrogen). For the Ikaros ChIP, 10 ul of a goat polyclonal Ikaros antibody ((M-20), Santa Cruz)) was diluted in 1 ml immune complex buffer with Dynabeads protein G (Invitrogen). 200 mg chromatin DNA was immunoprecipitated with antibody-bead complexes at 4oC overnight. The chromatin-DNA complexes were washed with immune complex buffers (Low salt, High salt, LiCl and TE) and were eluted followed by reverse cross-linking (10 mM Tris-HCl, 300 mM NaCl, 55 mM EDTA, 0.5% SDS). Sequencing libraries were generated using Illumina TruSeq V3 protocol and subjected to 51 bp single-end sequencing on an Illumina HiSeq2000.
attributes.tag: GEO Accession
attributes.value: GSM2458154