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 nine official subjects, which are the same in the Choice.

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

 IMPORTANT !!!

Please do not make extensive cut-and-paste: it s useless, anybody can go to the source you use and read it.  Read the texts, digest, and make a short résumé. If you wih you can include link(s) to the source(s).

Other contributors can revise, add, erase, modify...   Please do not repeat the same text as well. 


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

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Modified: 31 March 2020, 11:01 AM   User: Marianna Saviozzi  → 

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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 this 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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ATAC-Seq

(Marianna Saviozzi)

Assay for Transposase Accessible Chromatin with high throughput sequencing is a method for mapping CHROMATIN ACCESSIBILITY genome-wide. It makes use of an hyperactive version of the bacterial Tn5 transposase pre-loaded with sequencing adapters, that are inserted into accessible regions of chromatin. In physiological conditions Tn5 transfers a DNA fragment from a genomic latation to another: in this application it is pre-loaded with 2 sequencing adapters therefore their insertion into the accessible chromatin regions leads to genome fragmentation (tagmentation). These fragments are then PCR amplified and sequenced by using NGS technologies. The sequencing peacks correspond to open chromatin since sequencing starts from the accessible sites where Tn5 has inserted the adapters.

Pros:

  • Fast, simple and sensitive approach (preparation can be completed in 3 hours)
  • Works with many cell types and species
  • Requires no sonication, phenol-chlorophorm extraction (FAIRE), or antibodies  (ChIP-Seq)
  • Modifications have been made to the protocol in order to perform single-cell analysis.

Cons:

  • The number of cells must be optimized from the beginning: too few cells leads to under-transposition while too many leads to over-transposition (for studies on human cells 500-50000 cells are recommended but the optimal number may vary according to cell type and species)

Applications:

  • Nucleosome mapping: identification of changes in nucleosome position during differentiation or between experimental conditions, and correlation with sequence context.
  • Transcription factors occupancy analysis: information complementary to FAIRE ans Dnase-Seq outputs.
  • Identification of novel enhancers during development.
  • Deep study of the genomic prophile associated to pathological conditions such as cancer

  • In this panel ATAC-Seq workflow is described.
















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