Sequential Chromatin Immunoprecipitation to Identify Heterotypic Nucleosomes

Abstract

Sequential ChIP (ChIP-reChIP) enables the characterization of the same nucleosome for two different types of modifications or histone subtypes. Here, we describe a ChIP-reChIP protocol to identify a heterotypic (asymmetric) H2A.Z–H2A-containing nucleosome. In this method, following MNase digestion of chromatin to mostly a mononucleosome fraction, H2A.Z-containing nucleosomes are first immunoprecipitated using affinity purified anti-H2A.Z antibodies. This H2A.Z-containing nucleosome fraction is then subsequently immunoprecipitated using anti-H2A affinity purified antibodies to yield an enriched population of heterotypic H2A.Z–H2A containing nucleosomes. This protocol can be adopted to investigate any pair-wise combination of any histone variant, histone posttranslational modification, or any other protein that binds to a modified nucleosome. Show more