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