GSM5550774: RAW_E4BP4_rep2; Mus musculus; ChIP-Seq
RAW264.7 cells (54 million) were fixed for 30 minutes with 2 mM disuccinimidyl glutarate (DSG) and for 10 minutes with 1% formaldehyde. Nuclei were then isolated in buffer containing 1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.0, and protease inhibitors and sonicated using a Bioruptor (COSMO BIO) to shear chromatin to 200-1000 bp fragments. Protein-DNA complexes were incubated with two kinds of antibodies against E4BP4 (sc-28203; Santa Cruz Biotechnology and 11773-1-AP; Proteintech) and immunoprecipitated with IgG paramagnetic beads (Invitrogen). Eluted chromatin was isolated using MinElute PCR Purification Kit (QIAGEN). Sequencing libraries were generated using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) according to manufacturer's instructions. Library concentrations were assessed by both a Bioanalyzer (Agilent) and NEBNext Library Quant Kit for Illumina (New England Biolabs).