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Epigenomics: ChIP-Seq, DNase-Seq, FAIRE, ATAC-Seq, Nucleosome positioning
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Modified: 31 March 2020, 10:58 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
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:
- DNA-protein complexes are treated with DNase I;
- DNA extraction and sequencing are perfomed;
- Sequences bound by regulatory proteins are protected from DNase I digestion;
- 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:
And this is a video made by a Biology Professor at Davidson College, it explains the protocol in really easy terms:
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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
