GSE110681
Simplified ChIP-exo assays
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. Overall design: Several versions of optimized ChIP-exo and ChIP-seq protocols are described below: ChIP-exo 1.1 libraries were prepared for sequencing using standard Illumina protocols with a minor modification; Reference paper: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813302/ ChIP-exo 1.1 Low Chromatin libraries were prepared for sequencing using standard Illumina protocols with a minor modification; Reference paper: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813302/, but used 5 times less starting material was used. ChIP-exo 3.0 libraries were prepared for sequencing using standard Illumina protocols, Reference paper: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813302/, with modifications to include a Tn5 transposase to add adapters via tagmentation. Following tagmentation, the ChIP-exo 1.1 protocol was followed. ChIP-exo 3.0 Nextera libraries were prepared as ChIP-exo 3.0, but the in-house Tn5 was replaced by the protein that is commercially available from Illumina. ChIP-exo 4.0 libraries were prepared for sequencing using standard Illumina protocols, Reference paper: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813302/, with modifications to the method of adapter ligation. ChIP-exo 4.1 libraries were prepared for sequencing using standard Illumina protocols, Reference paper: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813302/, with modifications to the method of adapter ligation. that were opposite polarity of those used in ChIP-exo 4.0. ChIP-exo 5.0 libraries were prepared for sequencing using standard Illumina protocols, Reference paper: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813302/ with modifications throughout the protocol that reduced the number of steps and incubation times compared to ChIP-exo 1.1. ChIP-exo 5.0 Low Chromatin Low Chromatin libraries prepared for sequencing as for ChIP-exo 5.0, but used 5 times less starting material was used. ChIP-nexus (ChIP-exo 2.0) libraries were prepared for sequencing using standard Illumina protocols. Reference paper: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4390430/ ChIP-seq 1-step (altChIP-seq) libraries were prepared for sequencing using standard Illumina protocols with modifications that removed standard end repair steps and ligated the adapters in a single step. ChIPmentation (altChIP-seq) libraries were prepared for sequencing using standard Illumina protocols; Reference paper: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4589892/