GSM8254511: HOXC4_Input; Mus musculus; ChIP-Seq
Primary brown adipocytes overexpressing HOXC4 were fixed with 1% formaldehyde for 10 min at room temperature to cross-link DNA-protein complexes. Glycine was added to a final concentration of 125 mM for 5 min to terminate the crosslinking reaction. Cells were washed with PBS, harvested, and centrifuged at 900 × g for 3 min. The pellet was lysed to extract nucleus by centrifuging at 2000g for 5min. Genomic DNA was sonicated to 200 bp to 500 bp fragments. Of the sonicated chromatin, 10% was saved as the input sample, 80% underwent immunoprecipitation using anti-HA beads (MCE), and the remaining 10% was treated with rabbit IgG (Cell Signaling Technology) as the negative control. The DNA of input and IP was extracted by phenol-chloroform method. The high-throughput DNA sequencing libraries were prepared by using VAHTS Universal DNA Library Prep Kit for Illumina V3（Catalog NO. ND607, Vazyme）. The library products corresponding to 200-500 bps were enriched, quantified and finally sequenced on Novaseq 6000 sequencer (Illumina) with PE150 model.