Chromatin immunoprecipitation and multiplex sequencing (ChIP-Seq) to identify global transcription factor binding sites in the nematode Caenorhabditis elegans

Abstract

The global identification of transcription factor (TF) binding sites is a critical step in the elucidation of the functional elements of the genome. Several methods have been developed that map TF binding in human cells, yeast, and other model organisms. These methods make use of chromatin immunoprecipitation, or ChIP, and take advantage of the fact that formaldehyde fixation of living cells can be used to cross-link DNA sequences to the TFs that bind them in vivo. In ChIP, the cross-linked TF-DNA complexes are sheared by sonication, size fractionated, and incubated with antibody specific to the TF of interest to generate a library of TF-bound DNA sequences. ChIP-chip was the first technology developed to globally identify TF-bound DNA sequences and involves subsequent hybridization of the ChIP DNA to oligonucleotide microarrays. However, ChIP-chip proved to be costly, labor-intensive, and limited by the fixed number of probes available on the microarray chip. ChIP-Seq combines ChIP with massively parallel high-throughput sequencing (see Explanatory Chapter: Next Generation Sequencing) and has demonstrated vast improvement over ChIP-chip with respect to time and cost, signal-to-noise ratio, and resolution. In particular, multiplex sequencing can be used to achieve a higher throughput in ChIP-Seq analyses involving organisms with genomes of lower complexity than that of human (Lefrançois et al., 2009) and thereby reduce the cost and amount of time needed for each result. The multiplex ChIP-Seq method described in this section has been developed for Caenorhabditis elegans, but is easily adaptable for other organisms.

Keywords: Annealing protocol; Barcode sequences; Chromatin immunoprecipitation (ChIP); Formaldehyde-fixed Caenorhabditis elegans; Human Genome Project (HGP); Illumina Genome Analyzer; PCR primer preparation; Transcription factor (TF).