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Discover more at www.abcam.com Copyright © 2014 Abcam plc. Discover more at www.abcam.com
Applications of ChIP
November 05, 2014
David Grotsky, PhD
Scientific Support Specialist - Epigenetics
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Overview
Introduction to chromatin and ChIP
The histone code hypothesis
What we learn from ChIP-on-chip
What we learn from ChIP-seq
Variations of ChIP
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What is chromatin?
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Histone modifications
• ac – acetylation
• me – methylation
• P – phosphorylation
• cit – deimination
• ub – ubiquitination
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ChIP overview
Cross-link DNA and proteins (X-ChIP)
Chromatin fragmentation by sonication
(X-ChIP) or by enzymatic methods
(micrococcal nuclease) (N-ChIP)
Immunoprecipitation of the chromatin
fragments interacting with the target
protein/modification
Reverse cross-link (X-ChIP) and DNA
purification
Analysis of obtained material to determine
abundance of target sequence(s) relative to
input
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Downstream analysis
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An early link between histone acetylation
and transcriptional activation
Source: Hebbes et el, EMBO J, 1988
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An early link between histone acetylation
and transcriptional activation
Source: Hebbes et el, EMBO J, 1988
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The histone code hypothesis
Source: Strahl and Allis, Nature, 2000
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Common methods of ChIP analysis
ChIP-qPCR
ChIP-on-chip
ChIP-seq
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An example of ChIP-qPCR
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ChIP analysis genome-wide
Chip-on-chip
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Acetylation and methylation genome wide
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Source: Bernstein et al., PNAS, 2002
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ChIP-on-chip validation
Source: Bernstein et al., PNAS, 2002
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Using ChIP-on-chip to identify promoters
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Source: Kim et al., Nature, 2005
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Using ChIP-on-chip to identify promoters
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Source: Kim et al., Nature, 2005
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Using ChIP-on-chip to identify promoters
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Source: Kim et al., Nature, 2005
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Histone modification map of promoters
Source: Heintzman et al., Nature Genetics, 2007
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Histone modification map of enhancers
Source: Heintzman et al., Nature Genetics, 2007
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Limitations of ChIP-on-chip
Source: Bernstein et al., Cell, 2007
Requires large cell numbers
Not sensitive to repetitive elements
Large number of arrays are necessary to cover
entire genome
Amplification bias
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ChIP-seq
Source: Barski et al., Cell, 2007
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Higher resolution with ChIP-seq
Source: Park, Nature Reviews Genetics, 2009
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Higher resolution histone modification
map
Source: Barski et al., Cell, 2007
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Higher resolution histone modification
map
Source: Barski et al., Cell, 2007
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‘Dashboard’ of histone modifications
Source: Zhou et al., Nature Reviews Genetics, 2011
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Histone modifications on an active gene
Source: Barth and Imhof, Trends in Biochemical Sciences, 2010
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The ENCODE Project
ENCyclopedia Of DNA Elements
Identify all functional elements in the human genome
UCSC Genome Browser:
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ChIP-seq guidelines
Source: Landt, et al., Genome Research, 2012
Antibody validation
Sequencing depth
Experimental reproducibility
Data quality control
Data reporting
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Variations of ChIP
ChIP with low cell numbers
ChIP in under 5 hours
ChIP-loop
• Adds an IP step to chromatin conformation capture (3C) identifies long range
DNA interactions
• Detects DNA-DNA interactions mediated by a specific protein of interest
ChIA-PET (Chromatin Interaction Analysis by Paired-End Tag Sequencing)
• Genome wide ChIP-loop
• During ligation, linker sequences are added to create a library
of interacting regions
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Variations of ChIP
• Detects protein binding at single-nucleotide resolution
• DNA not bound directly by protein is removed
• PCR adapters are added to ChIP’d DNA and DNA is digested a
5’3’ exonuclease
• Crosslinks are reversed and DNA is amplified using primers to the
first adapter
• Second adapter is added to 5’ ends and product is sequenced
ChIP-exo
• Combination of ChIP and bisulfite sequencing
• ChIP products are bisulfite treated and then sequenced
ChIP-BS-
seq
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Products for your research
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Characteristics of abcam ChIP kits
Simple to
use
Reaction takes place in the wells of the 96-wp: easy for
standardization and HTP
Quick Only 5 hours to perform ChIP
(compared to 2 days using conventional method)
Inclusive Kit contains all necessary reagents for ChIP reaction
(excluding cross-linking step)
Eluted DNA can be processed straight away by any DNA-
amplification methodCompatible
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1
10
100
1000
10000
0 2,000 4,000 20,000 100,000 500,000
Non-Immune IgG RNA polymerase II antibody
New ChIP kit – High Sensitivity
(ab185913)
Relative enrichment fold
Cell number
• Low cell input:
2000 cells or
0.5mg of tissue per
prep
• Protocol time: 5hrs
(chromatin to
DNA)
• Convenient format:
96 well plate
format
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MeDIP (methylated DNA ChIP) Kits
Specific antibody to enrich 5-mC/5-hmC rich
DNA regions
• MeDIP – Detects 5-mC (methylated cytosine)
residues
• hMEDIP – Detects 5-hmC (hydroxymethylated
cytosine). 5-hmC is a recently discovered
modification in animal cells apparently involved
in DNA demethylation, but its role is not clear
Ready in less than 4 hours
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Chromatin Extraction Kit (ab117152)
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Source: Image courtesy of anonymous Abreview
Optimized kit to prepare chromatin for
ChIP experiments
Processing time < 1 hour
Sample: 105 cells or 10 mg tissue
Chromatin extracted from 3x106 mouse
neuroblastoma cells. Yield = 4 µg chromatin/106 cells
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Webinar promotion
As a
thank you
Save 20% on ChIP kits
• Hurry! This promotion is for a limited period only
• Simply quote the promo-code below at purchase
APPCHIP-XBLXZ
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Did you know Abcam has a technical
webinar series?
Have questions in regards to techniques?
• All of our past technical webinars are available online. Watch the webinars within your own
lab, on your time
www.abcam.com/webinars
Archived Webinars include
• ChIP
• ELISA
• In-Cell ELISA
• Western Blot
• Immunohistochemistry
• Immunocytochemistry
• Immunoprecipitation
• In-Cell ELISA
• Flow Cytometry
• Post Translations
Modifications
Archived Webinars include
Visit Abcam.com/events homepage for a full list of upcoming webinars and events
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Functions of Non-Coding RNAs in Evolution, Epigenetics and Therapeutic Applications
January 15, 2015 | Singapore
• Free meeting to attend | Early Registration Required
• Meeting organizers: Prof. Lawrence Stanton & Prof. Michael Rossbach, Genome Institute of
Singapore
Keynote Speaker: John Mattick, Garvan Institute, AU
"RNA at the epicenter of human development”
Confirmed Speakers
• Timothy Bredy, The University of Queensland, Brisbane, QLD,
Australia
• Roger Foo, National University Hospital, Singapore
• Michael Rossbach, GIS, Singapore
• Prabha Sampath, IMB, Singapore
• Lawrence Stanton, GIS, Singapore
• Leah Vardy, IMB, Singapore
• Wan Yue, GIS, Singapore
Speaker talk titles and more information at www.abcam.com/singapore2015
37
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Frontiers of Cell Signaling
June 21-24, 2015 | Shanghai, China
• Prof Yimin Zou, University of California - San Diego,US
• Yingzi Yang, NIH, US
• Dan Wu, Yale University, US
• Peter Lawrence, University of Cambridge, UK
• Andy McMahon, University of South California, US
• Susan Taylor, UCSD, US
• Early bird registrations: April 17, 2015 (save up to $325!)
• Oral abstract submission: April 17, 2015
• Standard registrations: May 7, 2015
• Poster abstract submission: May 7, 2015
Speaker talk titles and more
information at
www.abcam.com/cellsignaling
2015
38
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Questions?
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