index

ChIP-Seq:

ChIP-Seq combines chromatin immunoprecipitation with DNA sequencing, to obtain the DNA sequences which are able to interact with the immunoprecipitated proteins. This is very useful to identify transcription factors and nucleosome-associated sequences. Workflow is very simple: first, the cells need to be treated with formaldehyde, to create cross-link between the proteins and the DNA. Then, cells have to be lysed, DNA is extracted and fragmented with sonication; an antibody against the protein of interest is added. Precipitation could be achieved via beads associated to A/G proteins and centrifugation. Subsequently the pellet needs to be eluted and treated with high ionic force or high temperature, to resolve the cross-links and dissociate DNA fragments from the protein of interest. DNA has to be purified from contaminants (RNA and proteins). Finally, adapters have to be added at the ends of the fragments: ends must be blunted to allow ligase reaction between the fragments and the adapters. Specific primers for PCR phase (able to recognize these adapters) will be used to amplify and sequence each fragments. Also, a library can be created.

(Samuele Irudal)

ATAC-Seq

DNase-seq

DNase-seq (DNase I hypersensitive sites sequencing) is a technique used to identify the location of cis-regulatory regions (eg promoters, enhancers, silencers) in the genome. These regulatory areas are nucleosome-free and, therefore, are accessible and sensitive to DNase I. DNase I is an E. coli’s endonuclease which only shows specificity for DNA regions not wrapped on histones, independently from the nucleotidic composition.

Briefly, cells are lysed, chromatin in extracted and DNase I digestion occurr. Then the solution is deproteinized so only DNA fragments are cleaved by restriction enzymes. The remaining fragments are now separated on electrophoresis gel. In the classical DNase hypersensitivity assay the following step is Southern blot with very ends probes; on the contrary, in DNase-seq it is necessary to amplify cleavage products by PCR and prepare a library for high-throughput next generation sequencing. NGS will give the precise nucleotidic sequence of nucleosome-depleted DNA regions, which are the regulatory ones.

(Giada Cipollina)

FAIRE

Nucleosome positioning

other