Reconstitution and Purification of Nucleosomes with Recombinant Histones and Purified DNA

Abstract Nucleosomes are substrates for a broad range of factors, including those involved in transcription or chromosome maintenance/reorganization and enzymes that covalently modify histones. Given the heterogeneous nature of nucleosomes in vivo (i.e., varying histone composition, post‐translational modifications, DNA sequence register), understanding the specificity and activities of chromatin‐interacting factors has required in vitro studies using well‐defined nucleosome substrates. Here, we provide detailed methods for large‐scale PCR preparation of DNA, assembly of nucleosomes from purified DNA and histones, and purification of DNA and mononucleosomes. Such production of well‐defined nucleosomes for biochemical and biophysical studies is key for studying numerous proteins and protein complexes that bind and/or alter nucleosomes and for revealing inherent characteristics of nucleosomes. © 2020 Wiley Periodicals LLC. Basic Protocol 1 : Large‐scale PCR amplification of DNA Basic Protocol 2 : DNA and nucleosome purification using a Bio‐Rad Mini Prep Cell/Prep Cell Basic Protocol 3 : Nucleosome reconstitution via linear gradient salt dialysis