Antibodies you can trust : Unparalleled ChIP-Seq results withthe most rigorously validated antibodies
Jan Hendrickx, Géraldine Goens, Catherine D’Andrea, Mustafa Tammoh, Kana Sumikawa, Céline Sabatel, Miklos Laczik, Sharon Squazzo*, Hélène Pendeville, Dominique Poncelet
Diagenode sa, CHU, Tour GIGA B34, 3ème étage 1 Avenue de l’Hôpital, 4000 Liège, Sart-Tilman, Belgium I *Diagenode inc., 400 Morris Avenue, Suite 101, Denville, NJ 07834, USA

Introduction

Figure 2H3K4me3

Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of large
sets of genome-wide sequencing data. ChIP sequencing (ChIP-seq) has now become the gold standard
method for chromatin studies, given the accuracy and coverage scale of the approach over other methods.
Successful ChIP-seq, however, requires a higher level of experimental accuracy and consistency in all
steps of ChIP than ever before. Particularly crucial is the quality of ChIP antibodies.

Raw data from sequencers

In view of such requirements of highly qualified antibodies, Diagenode has established the most rigorousQC procedure for its new Premium Class of ChIP-seq grade antibodies. The Diagenode’s Premium
antibodies have reached the highest level of validation from the extensive work realized in numerous
collaborations and the IHEC community of epigenetic experts. All are validated for ChIP-seq and exhibit
superior performance for virtually any epigenetic application.

Checking the alignment andsequencing quality

The Premium antibodies passed the most stringent QC tests (including bioinformatics and comparison
with Encode reference data sets from the Broad Institute). Our partners consider these the best possible,
highest performance antibodies available and they will likely become the next international standards.
Our R&D work is in accordance with the overall objectives of the IHEC.
A guideline given by the IHEC consortium suggests to map the following 6 histone modifications within
any epigenome project: H3K4me3, H3K9me3, H3K27me3, H3K27ac, H3K4me1 and H3K36me3. The
presence of these marks is indicative of active promoters (H3K4me3, H3K27ac), active enhancers(H3K4me1, H3K27ac), actively transcribed genes (H3K36me3) or heterochromatin regions (H3K9me3,H3K27me3). Diagenode has followed these guidelines to generate specific antibodies recognizing thesehistone modifications.

Methods
Diagenode ChIP-seq workflow

1.

ELISA QC

H3K4me1

Read specific alignment

H3K36me3
ChIP-seq(Data kindly provided by theBlueprint consortium, JoostMartens, Willem Ouwehand& Henk Stunnenberg’s teams(Radboud University Nijmegen,
The Netherlands; University ofCambridge, UK)

H3K27me3

ChIP-seq Input DNA

H3K9me3

Antibody specific peak calling
H3K4me3

QC checks on peaks

Visual control, peak characteristics, signal to noise ratio, genome coverage,
genome feature affinity

H3K27ac

H3K4me1Comparison with reference
datasets

Comparing peak profiles, QC values to replicates, different lots, different
methods, published data (eg. Encode)

H3K36me3
H3K27me3

H3K9me3

Results

Peaks gained with Premium Antibodies matching peaks in Encode dataset

Chromatin stabilized
by cross-linking

Figure 3

Overlap of
all peaks
Overlap of
the top 40%
of peaks

These overlapping figures
are normally characteristic ofreplicate datasets.

ChIP process2.

Specificity/Cross reaction QCusing Bioruptor®

Broad H3K4me3

Diagenode H3K27ac

ChIP using the iDeal
ChIP-seq kit anda.

Assay Analysis

4.

Specificity/Cross reaction QC

Data Analysisb.

c.

As part of our philosophy to apply the highest quality standards to our antibodies and in an effort tocontinuously improve our QC procedure, we have introduced the validation of the Diagenode Premium
antibodies using highly stantardized ChIP-seq workflows.

d.

Broad H3K27ac

Diagenode H3K4me1

The Premium antibodies passed the most stringent QC tests (including general data analysis (eg. readsin peaks) and comparisons with the Encode reference data sets from the Broad Institute). Our partners
consider these as the best possible, highest performance antibodies available and they will likely become
the next international standards.

Sequencing library
preparation

5.

Broad H3K4me1

Diagenode H3K27me3

ChIP-seq run on Gallx
Illumina sequencer

Chromatin IPConclusions

3.

Premium antibodies

Affinity purification

Diagenode H3K4me3

Chromatin fragmentation

Chromatin IP

Diagenode’s Premium Antibodies

Broad H3K27me3

6.
Bioinformatics (mapping,

Diagenode H3K36me3

peak calling, statistics
and comparison)

Broad H3K36me3

ChIP-seq
Figure 1
H3K4me3, H3K27ac, H3K4me1, H3K36me3, H3K27me3 ChIP-seq profiles in K562 cells (Blue profile: Diagenode’s data;
Violet profile: Broad data).

www.diagenode.com

We used reference datasetsfrom the Encode project toconfirm the peaks detectedwith our Premium antibodies,direct matches were required(no distance between peaks),
and we achieved exceptional
overlap rates: the best 40%
of our peaks (likely containingno false peaks) often achievenearly 100% overlap ratio.

ProductsCat No.

ProductsCat No.

ProductsCat No.

H3K4me3 polyclonal
antibody - Premium

pAb-003-050

H3K4me1 polyclonal
antibody - Premium

pAb-194-050

H3K9/14ac polyclonal
antibody - Premium

C15410200

H3K36me3 polyclonal
pAb-192-050
antibody - Premium

H3K27me3 polyclonal
pAb-195-050
antibody - Premium

H2A.Zac polyclonal
antibody - Premium

C15410202

H3K9me3 polyclonal
antibody - Premium

H3K27ac polyclonal
antibody - Premium

H4K20me3 polyclonal
antibody - Premium

C15410207

pAb-193-050

pAb-196-050

For more information, please contact: for Europe, info@diagenode.com & for the US and Canada, contact info.na@diagenode.com

PO-Prem_Abs-A3-V1_21_01_13

CRUDE SERUM

H3K27ac

ChIP-seq experiments were performed on different blood cell types (Figure 1: K562 - Diagenode;
Figure 2: neutrophils - Diagenode’s external partner). Typically, cells are grown under standard conditions,
harversted by trypsinization and fixed with formaldehyde for 8 min. at room temperature. Crosslinked cells
are then sonicated using the Bioruptor® and the chromatin is tested on an agarose gel and Bioanalyzer
for a proper fragment distribution check. Immunoprecipitation was performed on 100,000 cells using theiDeal ChIP-seq kit and ChIP-seq Premium antibodies, crosslinks were reversed and purified DNA wastested for specific enrichments at pre-defined genomic regions using qPCR. If necessary, the IP’d DNA
from different ChIPs is pooled and sequenced using standard or multiplexed library preparation, cluster
generation and sequencing on an Illumina GAIIx. The sequenced tags were mapped to hg18, processedfor visualization in the UCSC browser and checked using our bioinformatics QC workflow.

Diagenode’s antibody quality control and ChIP-seq workflows:
QC from crude sera to purified
ChIP-seq grade antibodies

H3K4me3, H3K27ac, H3K4me1,
H3K36me3, H3K27me3 andH3K9me3 ChIP-seq profiles
in neutrophils (bottom panel– chromosome 19 views for 2zoom-in levels