User:Hankwbass/sandbox/b

Workflow[edit]
 Wet-lab portion of the workflow:  Figure 1. Biotinylated universal linkers with Mme1 restriction endonuclease sites are introduced. Figure 2. Biotinylated universal linkers are ligated to the free DNA ends. Figure 3. Ligation of linkers during proximity ligation. Figure 4. Pull down of biotinylated linkers by streptavidin-beads, and amplification of DNA tags. Figure 5. Conformations of universal linkers.


 * Formaldehyde is used to cross-link the DNA-protein complexes. Sonication is used to break-up the chromatin and also to reduce non-specific interactions.
 * A specific antibody of choice is used to enrich protein-of-interest–bound chromatin fragments. ChIP material bound by the antibody are used to construct the ChIA-PET.
 * Figure 1. Biotinylated oligonucleotide half-linkers containing flanking MmeI sites are used to connect proximity ligated DNA fragments. Two different linkers are designed (A and B) with specific nucleotide barcodes (CG or AT) for each of the two linker sequences (this will allow the identification of the chimeric ligation product as described in Figure 5.).
 * Figure 2. The linkers are ligated to the tethered DNA fragments.
 * Figure 3. The linker fragments are ligated on the ChIP beads under dilute conditions. The purified DNA is then digested by MmeI, which cuts at a distance from its recognition site to release the tag-linker-tag structure.
 * Figure 4. The biotinylated PETs are then immobilized on streptavidin-conjugated magnetic beads.
 * Figure 5. PET sequences with AA (CG/CG) and BB (AT/AT) linker barcode composition are considered to be possible intra-complex ligation products, while the PET sequences with AB (CG/AT) linker composition are considered to be derived from chimeric ligation products between DNA fragments bounded in different chromatin complexes.

 Dry-lab portion of the workflow: 

PET extraction, mapping, and statistical analyses

The PET tags are extracted and mapped to the reference human genome in silico.

Identification of ChIP enriched peaks (binding sites)

Self-ligated PET are used for identifying ChIP enriched sites because they provide the most reliable mapping (20 + 20 bit/s) to the reference genome.

ChIP enrichment peak-finding algorithm

A called peak is considered a binding site if there are multiple overlapping self-ligated PETs. The false discovery rate (FDR) is determined using statistical simulations to estimate the random background of PET-derived virtual DNA overlaps, and the estimated background noise.

Filtering of repetitive DNA (affects non-specific binding)

Satellite regions and binding sites present in regions with severe structural variations are removed.

ChIP enrichment count

The numbers of self-ligation and inter-ligation PETs (within + 250 bp window) are reported at each site. The total number of self-ligated and inter-ligated PETs at a specific site is called the ChIP enrichment count.

'''Figure 6. PET Classification: Uniquely aligned PET sequences can be classified by whether they are derived from one DNA fragment or two DNA fragments.''' Figure 6. Intra and inter-ligated PETs are clustered around TFBS when mapped to the reference human genome.


 * Self-ligation PETs

If the two tags of a PET are mapped on the same chromosome with the genomic span in the range of ChIP DNA fragments (less than 3 Kb), with expected self-ligation orientation and on the same strand, they are considered to be derived from a self-ligation of a single ChIP DNA fragment, and considered a self-ligation PET.


 * Inter-ligation PETs

If a PET does not fit into these criteria, then the PET most likely resulted from a ligation product between two DNA fragments and referred to as an inter-ligation PET. The two tags of an inter-ligation PETs do not have fixed tag orientations, might not be found on the same strands, might have any genomic span, and might not map to the same chromosome.


 * Intrachromosomal inter-ligation PETs

If the two tags of an inter-ligation PET are mapped in the same chromosome but with a span > 3 Kb in any orientation, then these PETs are called intrachromosomal inter-ligation PETs.


 * Interchromosomal inter-ligation PETs

PETs which are mapped to different chromosomes are called interchromosomal inter-ligation PETs.

'''Figure 7. Proposed mechanism showing how distal regulatory elements can initiate long-range chromatin interactions involving promoter regions of target genes.''' Figure 7. Proposed DNA looping mechanism between distal regulatory proteins and the promoter region The interactions form DNA loop structures with multiple TFBS at the anchoring center. Small loops might package genes near the anchoring center in a tight sub-compartment, which could increase the local concentration of regulatory proteins for enhanced transcriptional activation. This mechanism might also enhance transcription efficiency, allowing RNA pol II to cycle the tight circular gene templates. The large interaction loops are more likely to link together distant genes at either end of the loop residing near anchor sites for coordinated regulation, or could separate genes in long loops to prevent their activation. Adapted from Fullwood et al. (2009).

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Workflow[edit]
 Wet-lab portion of the workflow:  Figure 1. Biotinylated universal linkers with Mme1 restriction endonuclease sites are introduced.

Figure 2. Biotinylated universal linkers are ligated to the free DNA ends. Figure 3. Ligation of linkers during proximity ligation. Figure 4. Pull down of biotinylated linkers by streptavidin-beads, and amplification of DNA tags. Figure 5. Conformations of universal linkers.


 * Formaldehyde is used to cross-link the DNA-protein complexes. Sonication is used to break-up the chromatin and also to reduce non-specific interactions.
 * A specific antibody of choice is used to enrich protein-of-interest–bound chromatin fragments. ChIP material bound by the antibody are used to construct the ChIA-PET.
 * Figure 1. Biotinylated oligonucleotide half-linkers containing flanking MmeI sites are used to connect proximity ligated DNA fragments. Two different linkers are designed (A and B) with specific nucleotide barcodes (CG or AT) for each of the two linker sequences (this will allow the identification of the chimeric ligation product as described in Figure 5.).
 * Figure 2. The linkers are ligated to the tethered DNA fragments.
 * Figure 3. The linker fragments are ligated on the ChIP beads under dilute conditions. The purified DNA is then digested by MmeI, which cuts at a distance from its recognition site to release the tag-linker-tag structure.
 * Figure 4. The biotinylated PETs are then immobilized on streptavidin-conjugated magnetic beads.
 * Figure 5. PET sequences with AA (CG/CG) and BB (AT/AT) linker barcode composition are considered to be possible intra-complex ligation products, while the PET sequences with AB (CG/AT) linker composition are considered to be derived from chimeric ligation products between DNA fragments bounded in different chromatin complexes.

 Dry-lab portion of the workflow: 

PET extraction, mapping, and statistical analyses

The PET tags are extracted and mapped to the reference human genome in silico.

Identification of ChIP enriched peaks (binding sites)

Self-ligated PET are used for identifying ChIP enriched sites because they provide the most reliable mapping (20 + 20 bit/s) to the reference genome.

ChIP enrichment peak-finding algorithm

A called peak is considered a binding site if there are multiple overlapping self-ligated PETs. The false discovery rate (FDR) is determined using statistical simulations to estimate the random background of PET-derived virtual DNA overlaps, and the estimated background noise.

Filtering of repetitive DNA (affects non-specific binding)

Satellite regions and binding sites present in regions with severe structural variations are removed.

ChIP enrichment count

The numbers of self-ligation and inter-ligation PETs (within + 250 bp window) are reported at each site. The total number of self-ligated and inter-ligated PETs at a specific site is called the ChIP enrichment count.

'''Figure 6. PET Classification: Uniquely aligned PET sequences can be classified by whether they are derived from one DNA fragment or two DNA fragments.''' Figure 6. Intra and inter-ligated PETs are clustered around TFBS when mapped to the reference human genome.


 * Self-ligation PETs

If the two tags of a PET are mapped on the same chromosome with the genomic span in the range of ChIP DNA fragments (less than 3 Kb), with expected self-ligation orientation and on the same strand, they are considered to be derived from a self-ligation of a single ChIP DNA fragment, and considered a self-ligation PET.


 * Inter-ligation PETs

If a PET does not fit into these criteria, then the PET most likely resulted from a ligation product between two DNA fragments and referred to as an inter-ligation PET. The two tags of an inter-ligation PETs do not have fixed tag orientations, might not be found on the same strands, might have any genomic span, and might not map to the same chromosome.


 * Intrachromosomal inter-ligation PETs

If the two tags of an inter-ligation PET are mapped in the same chromosome but with a span > 3 Kb in any orientation, then these PETs are called intrachromosomal inter-ligation PETs.


 * Interchromosomal inter-ligation PETs

PETs which are mapped to different chromosomes are called interchromosomal inter-ligation PETs.

'''Figure 7. Proposed mechanism showing how distal regulatory elements can initiate long-range chromatin interactions involving promoter regions of target genes.''' Figure 7. Proposed DNA looping mechanism between distal regulatory proteins and the promoter region The interactions form DNA loop structures with multiple TFBS at the anchoring center. Small loops might package genes near the anchoring center in a tight sub-compartment, which could increase the local concentration of regulatory proteins for enhanced transcriptional activation. This mechanism might also enhance transcription efficiency, allowing RNA pol II to cycle the tight circular gene templates. The large interaction loops are more likely to link together distant genes at either end of the loop residing near anchor sites for coordinated regulation, or could separate genes in long loops to prevent their activation. Adapted from Fullwood et al. (2009).