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.


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

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ChIP-Seq

(Cecilia Boretto)

ChIP-Seq identifies the binding sites of DNA-associated proteins and can be used to map global binding sites for a given protein.

ChIP-seq protocol:

  • the first stage is to stabilize the link between proteins and DNA (DNA-protein crosslinking) thanks to formaldehyde;
  • wash and collect the cells with PBS follows;
  • the second stage is to fragment the DNA (with the bound proteins) thanks to a process known as "sonication" in lisis buffer (sonication occurs in different sonication and pause cycles, usually 12, in order to avoid the formation of foam that could escape from the eppendorf causing sample loss);
  • the third stage consists in the addition of a specific antibody for the protein of interest, the antibody is linked to a beads (sepharose or magnetic beads) which, thanks to its weight, deposits the antibody-protein-DNA complex on the bottom of the eppendorf;
  • the complexes are then collected and purified from non-specific proteins;
  • the last stage is the removal of the DNA protein bond thanks to the protease K;
  • the extracted DNA fractions can then be sequenced with NGS
  • after sequencing these can be aligned to the genome
  • after the alignment the peak is identified

Advantage:

  • ChIP-Seq does not require prior knowledge
  • ChIP-Seq delivers genome-wide profiling with massively parallel sequencing, generating millions of counts across multiple samples for cost-effective, precise, unbiased investigation of epigenetic patterns
  • Captures DNA targets for transcription factors or histone modifications across the entire genome of any organism
  • Defines transcription factor binding sites
  • Reveals gene regulatory networks in combination with RNA sequencing and methylation analysis 
  • Offers compatibility with various input DNA samples

Disadvantage:

  • Large Scale assays using ChIP is challenging using intact model organisms. This is because antibodies have to be generated for each TF, or, alternatively, transgenic model organisms expressing epitope-tagged TFs need to be produced
  • Researchers studying differential gene expression patterns in small organisms also face problems as genes expressed at low levels, in a small number of cells, in narrow time window
  • ChIP experiments cannot discriminate between different TF isoforms (Protein isoform)


More informations can be found here: 

DNase-Seq

(Emilia Petrachi)

DNase-Seq is one of the several approaches in molecular biology useful to identify DNA response elements, or regulatory regions in general, through genome-wide sequencing of regions sensitive to cleavage by DNase I.
A brief outline of the technique is the following:

  1. DNA-protein complexes are treated with DNase I;
  2. DNA extraction and sequencing are perfomed;
  3. Sequences bound by regulatory proteins are protected  from DNase I digestion;
  4. Deep sequencing is performed to provide accurate representation of location of regulatory proteins in the genome.

Pros

  • Can detect open chromatin
  • No prior knowledge of the sequence or binding protein is required
  • Compared to formaldehyde-assisted isolation of regulatory elements and sequencing (FAIRE-seq), has greater sensitivity at promoters

Cons

  • DNase l is sequence-specific and hypersensitive sites might not account for the entire genome
  • DNA loss through the multiple purification steps limits sensitivity
  • Integration of DNase I with ChIP data is necessary to identify and differentiate similar protein-binding sites

More information can be found at the website:
https://emea.illumina.com/science/sequencing-method-explorer/kits-and-arrays/dnase-seq-dnasel-seq.html
And this is a video made by a Biology Professor at Davidson College, it explains the protocol in really easy terms:

Brief outline of the DNase-Seq protocol

Another outline of the protocol

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