Chromatin immunoprecipitation followed by sequencing is used to detect DNA regions bound to a protein target by cross-linking proteins to bound DNA, breaking the unbound DNA (e.g. by sonication), extracting the protein target using an antibody, and then separating and sequencing the bound DNA fragments.
A modification of ChIP-seq was developed (Tagmentation-Assisted Fragmentation ChIP) to allow its application to small cell populations [1]. Tagmentation, the random fragmentation of DNA using the transposase Tn5, is used instead of sonication, resulting in a protocol with fewer steps, which prevents loss of material and reduces technical variability. The results of this new technique (e.g. on samples of 100 cells) compare well with those obtained with the classical ChIP-seq or with other variations specific for low cell input.
ChIP-seq results are often obtained with replicates. It is therefore possible to define variable occupancy target regions (VOTs) in genomes, by looking for regions producing not fully replicated binding for multiple targets. We developed a protocol to detect cell-specific VOTs using ChIP-seq data with replicates for the same cell type and for a few different transcription factors [2]. Application of the method to human cell lines K562, GM12878, HepG2, MCF-7, and in mouse embryonic stem cells (mESCs), found VOTs that are CG dinucleotide rich, and are enriched at promoters and R-loops. Conservation mESC VOTs in placental organisms and enrichment near DNA-binding genes suggest that VOTs reflect functional regions with highly dynamic interactions, possibly feedback loops of the gene regulatory network involved in development.
References
[1] Akhtar, J., P. More, S. Albrecht, F. Marini, W. Kaiser, A. Kulkarni, L. Wojnowski, J.F. Fontaine, M.A. Andrade-Navarro, M. Silies, C. Berger. 2019. TAF-ChIP: An ultra-low input approach for genome wide chromatin immunoprecipitation assay. Life Science Alliance. 2, e201900318.
[2] Andreani, T., S. Albrecht, J.F. Fontaine and M.A. Andrade-Navarro. 2020. Computational identification of cell-specific variable regions in ChIP-seq data. Nucleic Acids Research. 48, e53.