DNA-binding proteins, such as polymerases, transcription factors and chromatin modifiers, are critical for a variety of essential cellular processes, from DNA replication and repair to control of gene expression and chromatin structure. Individual DNA-binding proteins may bind to specific genomic loci, such as replication origins or promoters, or may bind DNA with varying sequence dependence, such as single-stranded DNA binding proteins, helicases, or polymerases (Farnham 2009; Johnson et al. 2007; Martin and Zhang 2005). Of primary concern to many researchers is the role that the location and modification of histones and nucleosomes play in gene regulation (Barski et al. 2007). Tri-methylation of histone 3 at lysine 4 has been shown to promote increased transcription of bound loci, while tri-methylation of histone 3 at lysine 27 results in decreased transcription (Martin and Zhang 2005). Chromatin immunoprecipitation (ChIP) has become an invaluable tool for studying the interactions between DNA-binding proteins and their genomic targets. Recent developments in next-generation sequencing (NGS) technologies have allowed scientists to more readily determine the target sequences of DNA-binding proteins on a genome-wide scale using ChIP coupled to high-throughput DNA sequencing (ChIP-seq).
The general workflow for a ChIP-seq experiment requires crosslinking proteins to DNA using a reversible cross-linker such as formaldehyde, cleaving the DNA by sonication or enzymatically, precipitating the protein-DNA complex of interest using specific antibodies coupled to magnetic beads, reversing the crosslink, preparing the library with the released DNA, and finally, high-throughput sequencing (Figure 1). By its very nature, the amount of DNA recovered from ChIP is very low, as only regions bound by a single protein or complex are selected. Moreover, ChIP from a limited number of cells poses an extra challenge. For those samples, the implementation of SMARTer ThruPLEX technology, which is designed for sample inputs as low as 50 pg, is key.