Project description:Genome-wide binding profile of the primate-specific KRAB zinc finger proteins ZNF676 and ZNF728 in cell lines overexpressing the proteins.
Project description:By combining chromatin immunoprecipitation (ChIP) with an exonuclease that digests the ends of protein-bound DNA fragments, ChIP-exo characterizes genome-wide protein-DNA interactions at near base-pair resolution. However, the widespread adoption of ChIP-exo has been hindered by several technical challenges, including lengthy protocols, the need for multiple custom reactions, and incompatibilities with recent Illumina sequencing platforms. To address these barriers, we systematically optimized and adapted the ChIP-exo library construction protocol for the unique requirements of mammalian cells and current sequencing technologies. We introduce a Mammalian-Optimized ChIP-exo (MO-ChIP-exo) protocol that builds upon previous ChIP-exo protocols with systematic optimization of crosslinking, harvesting, and library construction. We validate MO-ChIP-exo by comparing it to previously published ChIP-exo protocols and demonstrate its adaptability to both suspension (K562) and adherent (HepG2, mESC) cell lines. This improved protocol provides a more robust and efficient method for generating high-quality ChIP-exo libraries from mammalian cells.
Project description:Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and its ultra-high resolution cousin ChIP-exo are methods that identify where proteins bind along any genome in vivo. ChIP-exo achieves near-base pair resolution by creating exonuclease stop sites just 5’ to where formaldehyde-induced protein-DNA cross-links occur. Whereas construction of ChIP genomic libraries is straightforward and widely adopted for ChIP-seq, ChIP-exo is technically more involved which has resulted in limited adoption. Here we describe multiple ChIP-exo protocols, each with use-specific advantages and limitations. The new versions are greatly simplified through removal of multiple enzymatic steps. This is achieved in part through the use of Tn5 tagmentation and/or single-stranded DNA ligation. The result is greater library yields, lower processing time, and lower cost. A similar streamlined approach was developed for ChIP-seq, called ChIP-seq 1-step, where library construction is achieved in one-step.