Applications of ChIP - Abcam

Applications of ChIP

November 05, 2014

David Grotsky, PhD

Scientific Support Specialist - Epigenetics

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

What is chromatin?

Histone modifications

• ac – acetylation

• me – methylation

• P – phosphorylation

• cit – deimination

• ub – ubiquitination

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

Downstream analysis

An early link between histone acetylation

and transcriptional activation

Source: Hebbes et el, EMBO J, 1988

An early link between histone acetylation

and transcriptional activation

Source: Hebbes et el, EMBO J, 1988

The histone code hypothesis

Source: Strahl and Allis, Nature, 2000

Common methods of ChIP analysis

ChIP-qPCR

ChIP-on-chip

ChIP-seq

An example of ChIP-qPCR

ChIP analysis genome-wide

Chip-on-chip

Acetylation and methylation genome wide

Source: Bernstein et al., PNAS, 2002

ChIP-on-chip validation

Source: Bernstein et al., PNAS, 2002

Using ChIP-on-chip to identify promoters

Source: Kim et al., Nature, 2005

Histone modification map of promoters

Source: Heintzman et al., Nature Genetics, 2007

Histone modification map of enhancers

Source: Heintzman et al., Nature Genetics, 2007

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

ChIP-seq

Source: Barski et al., Cell, 2007

Higher resolution with ChIP-seq

Source: Park, Nature Reviews Genetics, 2009

Higher resolution histone modification

‘Dashboard’ of histone modifications

Source: Zhou et al., Nature Reviews Genetics, 2011

Histone modifications on an active gene

Source: Barth and Imhof, Trends in Biochemical Sciences, 2010

The ENCODE Project

ENCyclopedia Of DNA Elements

Identify all functional elements in the human genome

UCSC Genome Browser:

ChIP-seq guidelines

Source: Landt, et al., Genome Research, 2012

Antibody validation

Sequencing depth

Experimental reproducibility

Data quality control

Data reporting

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

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-

Products for your research

Characteristics of abcam ChIP kits

Simple to

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

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

• Protocol time: 5hrs

(chromatin to

• Convenient format:

96 well plate

format

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

Chromatin Extraction Kit (ab117152)

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

Webinar promotion

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

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

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

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

Questions?

Applications of ChIP - Abcam

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