Introduction of ChIP 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

DNA: Deoxyribonucleic acid Chromatin Chromosome

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? Chromatin Immunoprecipitation Gene Genomic Histon Non-histon

Chromatin Immunoprecipitation What is ChIP-Seq? Chromatin Immunoprecipitation Sequencing

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.

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 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.

× 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

Genome-wide identification of Protein–DNA binding sites

vitamin D receptor binding

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