Introduction of Introduction of

ChIP SequencingChIP Sequencing

Yanliu Lu, Ph.D.

The Key Lab of Basic Pharmacology

Outline

Definition and mechanism

Classification

Workflow and data analysis

Applications

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

Chromatin

Chromosome

DNA: Deoxyribonucleic acid

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

Chromatin Immunoprecipitation

What is ChIP?

• Gene Genomic• Histon Non-histon

Chromatin Immunoprecipitation

What is ChIP-Seq?

Sequencing

NUCLEAR RECEPTORS TARGET GENES

CYP7A

CYP7A, Intestinal bile acid-binding protein.

RXR/PPAR

RXR/LXR

RXR/FXR (RIP14)

Acyl-CoA Oxidase, Bifunctional enzyme (hydratase, 3-hydroxyacyl-CoA dehydrogenase), Thiolase, Very long chain acyl-CoA dehydrogenase, long chain acyl-CoA dehydrogenase, short chain-specific 3-detoacyl-CoA thiolase, and long chanin acyl-CoA synthetaseCYP4A1Liver fatty acid binding protinApoA1-I, Apo AII, ApoCIII

ChIP-Seq

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: 20-30 min;Sonication VS MNase.

ChIP workflow

Check quality and quantity of fragment DNA

For q-PCR: 200-800 bp;For sequencing: 200-600 bp.

ChIP workflow

Library workflow

Sequence identity and sequence similarity

Identity:

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.

Similarity (positive):

The extent to which two (nucleotide or amino acid) sequences are invariant.

Homology

Identity

Similarity

A is 80% identical to B

A is 80% similar to B ×

Homology

A is 80% homologous 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

Genome-wide identification of Protein–DNA binding sites

vitamin D receptor binding