Enabling solutions for fast and cost-effective ChIP-sequencing using
Diagenode iDeal ChIP-seq kit and antibodies with the Ion Torrent PGM™ sequencer
Irina Panteleeva, Jan Hendrickx, Miklos Laczik, Sharon Squazzo, Hélène Pendeville and Dominique Poncelet
Diagenode sa, CHU, Tour GIGA B34, 3ème étage 1 Avenue de l’Hôpital, 4000 Liège, Sart-Tilman, Belgium & Diagenode Inc: 400 Morris Avenue, Suite 101, Denville, NJ 07834, USA

Introduction

2. Validation of ChIP: reliable results using Diagenode’s ChIP-seq grade H3K4me3 antibody,isotype control and sets of validated primers

ChIP-seq has become the gold standard for whole-genome mapping of protein-DNA interactions.
The generalized adoption of this technology is currently limited by 4 main technical hurdles.

To provide the most robust and versatile ChIP-seq, we optimized the key steps of the ChIP workflow:

Figure 2.
Specific enrichment on positive loci (GAPDH, EIF4A2, c-fos promoter
regions) comparing to no enrichment on negative loci (TSH2B
promoter region and Myoglobin exon 2) was detected by qPCR.

First, the reproducibility and biological relevance of DNA-associated protein landscapes dependon the specificity and performance of the antibodies in the context for which they are used. Second,
the ChIP-seq method requires optimized protocols ensuring high recovery and increased signal-tonoise ratio. Third, as an effort to reduce the cost per sample and improve reproducibility, the ChIPseq method should be compatible with automation. Finally, the economical and widespread use ofChIP-seq requires access to a fast and high value/quality next-generation sequencing platform.

Samples were prepared using the Diagenode iDeal ChIP-seq kit.
Diagenode ChIP-seq grade antibody against H3K4me3 and thecorresponding isotype control IgG were used for immunoprecipitation.
qPCR amplification was performed with sets of validated primers.

In this poster, we demonstrate the successful use of the Diagenode iDeal ChIP-seq kit (Cat. No. AB001-0024) for ChIP-seq with the Ion Torrent Personal Genome Machine™.

The iDeal ChIP-seq kit is designed specifically for ChIP-seq(1)

1. Cell fixation and DNA-protein cross-linking.
2. Cell lysis and chromatin shearing using the Bioruptor ®.
3. Binding of the antibody to beads and chromatin.
4. Magnetic immunoprecipitation.
5. Washes of immune complexes.
6. DNA purification and recovery of ChIP’d DNA.

(2)

Methods A real breakthrough in Epigenetics
Ion Torrent has partnered with Diagenode’s world-class upstream sonication and epigenetic
technologies.

3. Superior performance of the Diagenode iDeal ChIp-seq kit with the IPure DNA purification
module and reliable sequencing with the Ion Torrent PGMTM sequencer

The Bioruptor ® sonicator is recommended for mechanical DNA shearing for Ion Torrent PGM™
library preparation. Diagenode has created and validated the iDeal ChIP-seq kit for Ion Torrent
PGM™ sequencing. The iDeal ChIP-seq kit is available for purchase from the Ion Torrent Store.

a.

b.

c.

b.

a.

c.

b.

Diagenode and Ion Torrent share the same goal : to provide every sequencing lab a simple, efficientand cost-effective sequencing workflow for ChIP-seq.
PrepareGenomic
Library

IP buffer (3)Optimal conditions for antibody
bindingAffinity enrichment using ChIPseq grade Ab (3, 4)

Improved specificity, reliableresultsd.

c.

Washes and washing buffers (5)Higher signal-noise ratio, lessbackground in sequencing

DNA purification using the IPurekit (6)Improved consistency andmaximum sample recoveryd.

Related Products

PrepareChIP
Library

Description
PrepareTemplateon BeadBenefits
High fragmentation efficiency, easy
to set up, reproducible results

d.

(3)
a.

Optimized steps
Chromatin buffers and shearing
conditions (1, 2)

Figure 3.

Cat. No.

Description

Cat. No.

Bioruptor ® Standard

4465622

H3K9ac polyclonal antibody

pAb-004-050

iDeal ChIP-seq kit x24 for PGM

AB-PGM-0024

H3K9ac polyclonal antibody

pAb-177-050

IPure kit x100

AL-100-0100

H3K9/14ac polyclonal antibody

pAb-005-044

H3K4me3 polyclonal antibody

pAb-003-050

H3K27ac polyclonal antibody

pAb-174-050

H3K4me3 monoclonal antibody

MAb-152-050

H4K20me3 polyclonal antibody

pAb-057-050

H3K4me2 polyclonal antibody

pAb-035-050

Pol II monoclonal antibody

AC-055-100

H3K4me1 polyclonal antibody

pAb-037-050

ER monoclonal antibody

AC-066-100

H3K9me3 polyclonal antibody

pAb-056-050

TBP monoclonal antibody

MAb-002-100

H3K27me3 polyclonal antibody

pAb-069-050

RARA polyclonal antibody

CS-155-100

H3K36me3 polyclonal antibody

pAb-058-050

Human c-fos promoter primer air pp-1004-050/500

H3K36me3 polyclonal antibody

CS-058-100

Human TSH2B primer pairResults

H3K79me3 polyclonal antibody

pAb-068-050

Human myoglobin exon 2 primer pair pp-1006-050/500

1. Efficient and easy chromatin shearing using the Bioruptor ® and Shearing buffer iS1 from theiDeal ChIP-seq kitConclusions

Sequence onIon ChIPSignal
Processingand
Base Calling

Figure A: The iDeal ChIP-seq kit with the IPure DNA purifcation module provides lower noise (morereads in the peaks, less in the background) and higher specificity (more peaks found in the promoter
regions for the promoter specific H3K4me3 antibody – less false positives). The lower noise level
and the subsequent higher read accumulation tendency is demonstrated by a higher average foldenrichment ratio as well for the iDeal ChIP-seq kit with the IPure DNA purification module.
Figure B: Using the IPure DNA purification module with the iDeal ChIP-seq, kit we detected
approximately the same number of peaks as with the column-based purification kit from Competitor
Q from roughly 40% less reads, using the same H3K4me3 antibody. This is due to the more specificcaptured reads and the lower noise level maintained by the IPure unique technology. The filtered
duplicate read ratio was also lower, 4.0% compared to the Competitor’s 5.6%.
Figure C: Using another Massive Parallel Sequencing system and the iDeal ChIP-seq kit with same
antibody confirmed most of the peaks, further proving the high ratio of true positive hits.

ChIP-seq Bioinformatics
Peak calling
Integrated bioinformatics

MW500 bp

100 bpHela

4. ChIP-seq data acquired by PGM maintain high specificity.
A.

B.

Figure 1.

••

Chromatin from 1 million of Hela cells was sheared using the Bioruptor ® combinedwith the Bioruptor ® Water cooler (Cat No. BioAcc-cool) during 3 rounds of 10
cycles of 30 seconds “ON” / 30 seconds “OFF” at HIGH power setting (position H).
Diagenode 1.5 ml TPX tubes (Cat No. M-50001) were used for chromatin shearing.

Figure 4.
The high consistency of the iDeal ChIP-seq kit is proven by multiple sequencings on Ion Torrent
PGM™ and another Massive Parallel Sequencer (MPS-I).

•Samples were gently vortexed before and after performing each sonication round(rounds of 10 cycles), followed by a short centrifugation at 4°C to recover the sample
volume at the bottom of the tube. The sheared chromatin was then decross-linked
as described in the kit manual and analyzed by agarose gel electrophoresis.

Figure A shows a several hundred bp stretch along 12th chromosome, where a high similarity ofread distribution can be observed, despite the radically different characteristics of the sequencers.

•Figure B is a close capture focusing on the well-known GAPDH gene (see dotted rectangle). The
detailed view reveals that even the peak structure is similar.

•

www.diagenode.com

pp-1041-050/500

The Diagenode iDeal ChIP-seq kit is tailor-made for ChIP-seq.
Performance of the iDeal ChIP-seq kit is optimal with Diagenode’s ChIP-seq grade antibodies
and IPure Magnetic DNA Purification module.
Proven efficiency and robustness of the iDeal ChIP-seq kit with Ion Torrent PGM TM sequencing
platform.
E xtremely high reproducibility and high peak overlap with PGM using the iDeal ChIP-seq kit
starting from as low as 10ng ChIP’d DNA.
Compatible with automation using the Auto iDeal ChIP-seq kit using the SX-8G IP-Star ® andSX-8G IP-Star ® Compact Automated Systems (data not shown).
PO-iDeal-A3-V1_30_07_12

For more information, please contact Dominique.poncelet@diagenode.com • For Europe, contact orders@diagenode.com • For the US and Canada, contact orders.na@diagenode.com