The native ChIP protocol using chromatin fragmentation with micrococcal nuclease (MNase) was used as described in (50). During nuclei isolation, buffers were supplemented with protease inhibitors (0.1 mM benzamidine and 0.1 mM phenylmethylsulfonyl fluoride). Nuclei (150,000 to 200,000) of each cell type were used per ChIP reaction. Anti-H3K27ac antibody was from Active Motif (catalog #39133; lot #31814008; rabbit polyclonal, 3 μg per sample). The antibodies were prevalidated for specificity using a dot blot assay (AbSurance Histone H3 Antibody Specificity Array Kit, Millipore). ChIP-seq libraries were prepared with the NEBNext Ultra DNA Library Prep Kit (New England BioLabs). The resulting libraries were sequenced on a HiSeq 2500 instrument, using a paired-end 50-cycle protocol, to an average of ~40 million read pairs per sample. For each sample, a matching input control sample obtained from 1 ng of MNase-digested DNA was prepared and sequenced.

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



Q&A
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.