1. Introduction of ChIP Sequencing
Yanliu Lu, Ph.D.
The Key Lab of Basic Pharmacology

2. Outline Definition and mechanism Classification
Workflow and data analysis
Applications

3. Zwei Dinge erfüllen das Gemüt mit immer neuer und zunehmender Bewunderung und Ehrfurcht, je öfter und anhaltender sich das Nachdenken damit beschäftigt: Der bestirnte Himmel über mir, und das moralische Gesetz in mir ——Immanuel Kant

4. DNA: Deoxyribonucleic acid
Chromatin
Chromosome

5. Histon and Non-histon protein
Active or represse
Over expression or knock out
Dominant-negative mutation
Transcription factor assay
Electrophoretic mobility shift assay
DNase I footprinting

6. Chromatin Immunoprecipitation
What is ChIP?
Chromatin Immunoprecipitation
Gene Genomic
Histon Non-histon

7. Chromatin Immunoprecipitation
What is ChIP-Seq?
Chromatin Immunoprecipitation
Sequencing

8. ChIP-Seq NUCLEAR RECEPTORS TARGET GENES RXR/PPAR
Acyl-CoA Oxidase, Bifunctional enzyme
(hydratase, 3-hydroxyacyl-CoA dehydrogenase),
Thiolase, Very long chain acyl-CoA dehydrogenase,
long chain acyl-CoA dehydrogenase, s
hort chain-specific 3-detoacyl-CoA thiolase,
and long chanin acyl-CoA synthetase
CYP4A1
Liver fatty acid binding protin
ApoA1-I, Apo AII, ApoCIII
RXR/LXR
CYP7A
RXR/FXR (RIP14)
CYP7A
, Intestinal bile acid-binding protein.

9. X-ChIP and N-ChIP
DNA-binding proteins, histone modifications or nucleosomes;
Based on with or without crosslinking, ChIP is referred to as X-ChIP and N-ChIP;
Crosslinking time: min;
Sonication VS MNase.

10. ChIP workflow

11. Check quality and quantity of fragment DNA
For q-PCR: bp;
For sequencing: bp.

12. ChIP workflow

13. Library workflow

16. Sequence identity and sequence similarity
The extent to which two (nucleotide or amino acid) sequences are invariant.
Similarity (positive):
The extent to which nucleotide or protein sequences are related. The extent of similarity between two sequences can be based on percent sequence identity and/or conservation. In BLAST similarity refers to a positive matrix score.

17. × Homology A is 80% identical to B Identity Homology Similarity
A is 80% homologous to B ×
A is 80% similar to B
If your sequences are more than 100 amino acids long (or 100 nucleotides long), you can label proteins as “homologous” if 25 percent of the amino acids are identical, for DNA you will require at least 70 percent identity

18. Genome-wide identification of Protein–DNA binding sites

23. vitamin D receptor binding

31. Thanks!