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Il principio della ChIP: arricchimento selettivo della frazione di cromatina contenente una specifica proteina La ChIP può anche esser considerata una strategia di reversegenetics su scala genomica There are many options for keeping samples cool during sonication: •Use the pulse mode to reduce heat buildup. •Put samples on ice along with the pulse mode. Tip Depth / Foaming Issue P robes/tips must be submerged properly. I f the tip is not submerged enough the sample will foam or bubble. If the tip is too deep it will not circulate the sample effectively. Both conditions will end up with poor results. Foaming often occurs with samples volumes below 1ml. Foaming can also be caused when the amplitude setting is too high. - La qualità dell’anticorpo è fondamentale in questa fase - Deve riconoscere la proteina legata al DNA - Deve essere altamente specifico - Necessaria una pre-taratura per ridurre il legame aspecifico -Gli immunocomplessi sono poi precipitati con ProtG/A Sepharose (lega la regione Fc dell’anticorpo) - lavaggi stringenti riducono il background DNA-binding proteins are crosslinked to DNA with formaldehyde in vivo. Isolate the chromatin. Shear DNA along with bound proteins into small fragments. Bind antibodies specific to the DNA-binding protein to isolate the complex by precipitation. Reverse the cross-linking to release the DNA and digest the proteins. Use PCR to amplify specific DNA sequences to see if they were precipitated with the antibody. “ChIP” • If we have the “right” antibody, we can extract (“immunoprecipitate”) from living cells the protein of interest bound to the DNA • And - we can try to identify which were the DNA regions bound by the protein • Can be done for transcription factors • But can be done also for histones - and separately for each modification History: From ChIP-chip to ChIP-seq ChIP-chip (c.2000) • Resolution (30-100bp) • Coverage limited by sequences on the array • Cross-hybridization between probes and non-specific targets creates background noise Workflow of ChIP-Seq ChIP-seq overview DNA + bound protein Fragment DNA Sequence Map sequence tags to genome & identify peaks Adapted from slide set by: Stuart M. Brown, Ph.D., Center for Health Informatics & Bioinformatics, NYU School of Medicine Prepare sequencing library Immunoprecipitate Release DNA ChIP-seq Challenges: • • • • • • Millions of segments Mapping to genome Visualization Peak detection Data normalization … ChIP seq experiment In Nutshell •Protein cross-linked to DNA in vivo by treating cells with formaldehyde •Shear chromatin (sonication) •IP with specific antibody •Reverse cross-links, purify DNA •PCR amplification* •Identify sequences •Genome-wide association map *-unless using a single molecule sequencer ChIP-seq big picture • Combine high-throughput sequencing with Chromatin Immuno-precipitation to identify specific protein-DNA interactions genome-wide, including those of: • • • • • Transcription factors Histones (various types and modifications) RNA Polymerase (survey of transcription) DNA polymerase (investigate DNA replication) DNA repair enzymes • … or fragments of DNA that are modified (e.g. methylated)