This document summarizes the steps of a ChIP-Seq (Chromatin Immunoprecipitation Sequencing) assay. The key steps are: 1) cross-linking proteins to DNA, 2) fragmenting chromatin, 3) immunoprecipitating the DNA-protein complex using antibodies specific to the protein of interest, 4) purifying and analyzing the DNA. ChIP-Seq allows researchers to identify the genomic binding sites of transcription factors and histone modifications genome-wide.

1. ChIP-Seq
Immunoprecipitation
(Chromatin Immunoprecipitation Sequencing)
Presented by : Amira Moustafa

2. Chromatin immunoprecipitation
• it was considered one of the most popular methods to study protein-DNA
interactions and can be used , to identify the binding sites of transcription
factors ( activators or repressors) or to determine the distributions of
histones ( histone modifications) with specific post-translational modifications
throughout the genome.

3. CHIP- assay Steps
• Cross- Linking of protein to DNA
• Cell lysis
• Chromatin Fragmentation
• Immunoprecipitation using specific Anti-bodies
• DNA purification
• DNA analysis

4. CHIP- assay Steps

5. Cross-linking of protein to DNA : this step was performed using cross- linking
agent (e.g. 16% formaldehyde (W:V) (methanol free), glutaraldehyde,
disuccinimidyl glutarate [DSG](preferred) .
Chromatin fragmentation : The chromatin is fragmented or “sheared” to
mononucleosome sized fragments (150-300 bp), which is important for obtaining
high resolution sequencing data. Chromatin shearing is accomplished by
sonication or enzymatic digestion using micrococcal nuclease (MNase) and
monitored by gel (e.g. agarose) or capillary electrophoresis .
Immunoprecipitation using specific Anti-bodies : Selected Abs should be highly –
specific Abs and show highly effective binding with chromatin associated
proteins . The amount of primary antibody required for good signal in a ChIP is
usually 2–5 μg . The antibody is coupled to magnetic beads , and incubate at 4°C
for at least 12 h, with rotation such that the samples mix and the beads remain
suspended . the antibody-bound chromatin is isolated from bulk chromatin using
a magnet, followed by a series of stringent washes.

6. Separation of Magnetic Beads with Bound
Antibody and Protein-DNA Complexes

7. • DNA purification : This step needed a proteases enzymes to digest proteins . For
Purification of ChIP and Input samples a Zymo ChIP DNA Clean & Concentrator Kit
were used. Elute in 26 μl of Kit Elution Buffer.
• DNA quantification : 0.5 - 1 μl should be taken from each sample for quantification
of DNA concentration using a Nanodrop or Qubit fluorometer.
• Data analysis:
1. Amplification of DNA samples by qPCR.
2. The e ChIP-seq DNA libraries may be safely stored in the freezer until convenient.
3. Determine the concentration of the libraries in ng/mL using a Qubit fluorometer .
4. Estimate the mean library size in base pair (bp) by running a small amount (2 mL)
of the library on a Tape Station, Bio analyzer, or a 2% agarose/TBE gel pre-stained
with GelGreen.
5. Analysis of Data by Easeq software