index

[Audrey CHAMPIER]

                                    ChIP-Seq 

Methode used to analyse the interactions between proteins and DNA.

The technique consists in a chromatin  immunoprecipitation followed by a parallel DNA  sequencing in order to identify the binding sites of one protein on DNA. ( Before it, scientists used Chip on Chip : Chromatin immunoprecipitation + sequencing with  DNA -microarray technique )

Chromatin immunoprecipitation :   ( for more details see  : " Wet-Lab portion of the workflow in the following link" :  https://en.wikipedia.org/wiki/ChIP-on-chip#Workflow_of_a_ChIP-on-chip_experiment)

The protein of interest is first cross-linked with the  DNA site it binds,  thanks to formaldehyde for example.

Then the cells are lysed and the DNA , thanks to sonication, becomes small fragments.

After this , we introduce an antibody specific of  the protein that interests us.  This antibodies are bound to a surface and so we obtain,on this surface, all the sequence of DNA that bind the protein. In order to obtain only the DNA sequence, we reverse the cross-linking of DNA and proteins,  thanks to heat.

Sequencing:  

We use Parallel DNA sequencing method in order to obtain the sequence of all the fragments of DNA we just obtained with immunoprecipitation. 

For more informations see: 

https://en.wikipedia.org/wiki/ChIP-sequencing#ChIP

[Audrey CHAMPIER]

                                               DNase-Seq 

DNase seq is a method in biology used to identify the location of regulatory region on DNA.

For this we used DNase-I, an enzyme  ables to cleave specifics sequences ( nucleosome-depleted DNA that are easier to cleave than the other sequences). This sequences are markers of open chromatine and contains active  regulatory regions. We obtained fragments of DNA that we can purified and  sequenced ( thanks to parallel DNA sequencing methods )

Should be completed ;)

[Valentina Serra]

                                                ATAC-seq

ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) is a technology that can be used to obtain information about genome-wide chromatin accessibility. It is an emerging technique that is gaining popularity as it aids in a fast and sensitive analysis of the epigenome compared for example to the DNase-seq technology.

This method probes DNA accessibility using the hyperactive mutant Tn5 transposase which inserts sequencing adapters into accessible regions of the chromatin. The protocol is quite simple: after the lysis of the cells, the transposase complex fragments the genome and tags the resulting DNA with sequencing adapters. The tagged DNA fragments are then purified, amplified by PCR and sent for sequencing (using the linkers, already added by the enzyme, for the library preparation). More information can be found here: https://www.abcam.com/epigenetics/epigenetics-application-spotlight-atac-seq

The advantages of this technology are:

-       the simplicity of the library preparation protocol

-       very fast

-       sonication or phenol-chloroform extraction are not required as for the FAIRE-seq; no antibodies like ChIP-seq; and no sensitive enzymatic digestion like MNase-seq or DNase-seq

The disadvantages are:

-        during the mechanical sample processing, bound chromatin regions might open and be tagged by the transposase

-        only half of the molecules contain the adapters in the orientation required for PCR amplification