ChIA-PET (Chromatin Interaction Analysis by Paired-End Tag Sequencing)

Alternative names:
Description: A technique that combines the ability of ChIP to target specific proteins and the ability of
3C to detect long range chromatin interactions. Determines long range interactions mediated by a
chosen protein.
Procedure:
1. Crosslink proteins to DNA using formaldehyde.
2. Shear DNA by sonication.
3. Immunoprecipitate DNA bound to the protein of interest using an antibody.
4. Process immunoprecipitated DNA ends to create blunt ends, then ligate under dilute conditions.
5. Degrade proteins and then purify the ligated DNA fragments.
6. Amplify your DNA and perform PET sequencing.
Uses:
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Chromatin Conformation Capture

Alternative names: 3C
Description: A technique to identify distant interacting regions of chromosomal DNA; uses real time
PCR (a single/several loci method).
Procedure:
1. Crosslink DNA to proteins using formaldehyde.
2. Digest DNA using restriction enzymes (chosen to ideally digest your region of interest).
3. Ligate DNA under dilute conditions to promote intramolecular joinings.
4. Use PCR to investigate loci of interest.
Uses:
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ChIP (Chromatin Immunoprecipitation)

Description: Detects specific DNA sequences a protein binds to. Uses PCR to amplify
immunoprecipitated DNA.
Procedure:
1.
2.
3.
4.

Cross-link proteins to DNA using formaldehyde.

Pull your protein and attached DNA out of solution using an antibody to the protein.
Using primers homologous to DNA sequences of interest, perform PCR on pulled down DNA.
Compare abundances of PCR product from immunoprecipitated DNA to PCR product from
non-immunoprecipitated DNA to determine whether protein binds to the DNA sequence of
interest.

Uses: histone modifying enzymes (in vivo),

ChIP-Seq
Description: Chromatin immunoprecipitation followed by deep sequencing analysis of the pulled down
DNA.
Procedure:
1.
2.
3.
4.
5.
6.

Crosslink protein to DNA using formaldehyde

Shear DNA
Immunoprecipitate target protein and associated DNA
Reverse crosslink; purify DNA
Prepare DNA for sequencing
Sequence DNA libraries and map reads back to the genome

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DNAse I Protection Assay

Alternative names: Footprinting Assay
Description: Detect DNA binding and binding site by incubating protein with end-labeled DNA and
digesting with DNAse I. Binding will protect a region of DNA from DNAse digestion. Band-free
regions on a denaturing gel reflect protection and regions bound by protein.
Procedure:
1. Step 1
Uses:
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GRO-Seq
Alternative names: Global Run-On followed by deep Sequencing
Description: A technique for identifying genes being actively transcribed at a given moment in a
population of cells.
Procedure:
1. Isolate nuclei and add rNTPs, including bromo-UTP.
2. Incubate to allow engaged RNA polymerases to incorporate bromo-UTP into RNA.
3. Isolate RNA and immunoprecipitate with an antibody against bromo-UTP to pull down actively
transcribed RNA.
4. Ligate different adaptors to the 5' and 3' ends of the immunoprecipitated RNA.
5. Reverse transcribe cDNA from the ligated adaptors.
6. Use deep sequencing to map and quantify the cDNA.
Uses:
Images:

Hi-C
Alternative names: High-throughput Chromatin Conformation Capture
Description: A technique that identifies distant chromatin interactions in an unbiased, genome-wide
fashion using biotin labeling and deep sequencing.
Procedure:
1. Crosslink DNA and protein with formaldehyde.
2. Cut the DNA using restriction enzymes that leave single stranded overhangs.
3. Fill in the single stranded ends with biotin labeled nucleotides.
4. Ligate under dilute conditions to promote intramolecular joining of DNA ends.
5. Reverse crosslinks and purify biotin-labeled DNA.
6. Shear DNA and sequence using PET sequencing.
Uses:
Images:

Mass spectrometry
Description: A method for identifying proteins and their modification state by digesting proteins with
proteases, determining the masses of the fragments, and matching that data to protein sequence
databases.

MNase Seq (Micrococcal Nuclease Sequencing assay)

Uses: nucleosome positioning (mostly in vivo)

Nucleosome Mapping Assay

Description: Extensively digest DNA with MNase, and then purify the ~160 bp sized DNA. Use
paired-end deep sequencing to determine all of the protected, nucleosomal regions.
Alternative names: MNase Seq
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RNA-Seq
Description: Take a snapshot of RNA transcript levels within a cell.
Procedure:
1.
2.
3.
4.
5.

Isolate RNA from cells.

Make cDNA from your RNA using reverse transcriptase.
Shear your cDNA into smaller pieces.
Ligate adaptors to your cDNA fragments.
Use deep sequencing to map and quantify the cDNA sequences (which will be proportional to
the RNA that you isolated).

Images:

Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)

Description: A technique for measuring the amount of a particular RNA transcript present in a sample.
Hybridize DNA primers to RNA, extend with reverse transcriptase, then perform PCR (using a DNA
polymerase) to amplify resulting DNAs. Use primers homologous to the 3' end of the transcript to
target only complete RNAs.

Assay Name
Alternative names:
Description:
Procedure:
2. Step 1
Uses:
Images: