Chromatin immunoprecipitation–sequencing

Cells expressing S100A8-Flag, S100A9-Flag, or untagged protein were grown in the presence or absence of tamoxifen, washed once with room temperature PBS, and then fixed with protein-protein cross-linking solution [1.5 mM ethylene glycol-bis (EGS), 1.5 mM disuccinimidyl glutarate (DSG), and 1.5 mM disuccinimidyl suberate (DSS) in PBS] for 45 min at room temperature. Then, protein-DNA cross-linking with 1% formaldehyde (methanol free) was performed for 15 min at room temperature, and the reaction was quenched by 125 mM glycine. For Pol II ChIP, standard formaldehyde cross-linking was performed. After fixation, nuclei were isolated and dissolved in sonication buffer (10 mM tris-HCl at pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine, and protease inhibitor cocktail) for sonication and subsequent immunoprecipitation (Flag antibody was used for negative control or S100A8 or S100A9 ChIP; Pol II antibody, 8WG16, was used for Pol II ChIP). The sheared chromatin fragments were enriched around 200 to 300 bp. For ChIP and corresponding chromatin input samples, DNA was isolated and used to prepare libraries, which were sequenced on Illumina NextSeq 500 or HiSeq 2000. Other ChIP-seq (H3K4me3, H3K4me1, H3K9me3, H3K27me3, H3K27ac, and MYC) and DNase sequencing (DNase-seq) experiments mentioned in this study were performed in transformed or nontransformed cells as previously described (21, 51).

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.