Student Wiki on methodology

This Wiki is intended to collectively make the point on methodologies employed in research papers we analyze during the course. "Writers" are students who wish to contribute to a specific subject. Before contributing, please add your name in the "Writers group choice". When initiating a contribution, please indicate your name in brackets.


PLEASE:  DO NOT change the INDEX page !!!
This page contains the links to the seven official subjects, which are the same in the Choice.

To contribute, go to the right page by clicking on the description here in the index, then click EDIT and contribute. At the end, please save.

 



Chromatin: ChIP-Seq, DNase-Seq, FAIRE, ATAC-Seq, Nucleosome positioning

Viewing page version #6
(Restore this version) 

Modified: 14 March 2018, 5:08 PM   User: Samuele Irudal  → 

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 libraries can be created.

(Samuele Irudal)

ATAC-Seq

DNase-seq

FAIRE

Nucleosome positioning

other