RBest Workflow Practices
for ChIP-seq Analysis withSmall Samples
by Irina Panteleeva, Céline Sabatel, Wassim Lakhal, Jerome Kroonen, Miklos Laczik, Rini Saxena,
Agnieszka Zelisko-Schmidt, Benjamin Deist, Chava Pocernich & Steve Siembieda

Abstract:
Combined chromatin immunoprecipitation and next-generation sequencing (ChIP-seq) has
become the gold standard to investigate genome-wide epigenetic profiles. However, ChIP froma limited amount of cells has been a challenge. Diagenode and Advanced Analytical Technologies,
Inc. (AATI) provide the first complete and robust workflow solution for successful ChIP-seq fromsmall numbers of cells.
Diagenode’s Bioruptor® Pico,True MicroChIP Kit and theMicroPlex Library Preparation™ Kit efficiently preparechromatin from very low input amounts as these
combined solutions:• Allow reproducible shearing from low inputs ofchromatin using the Bioruptor® Pico
• Assure quality with a validated ChIP protocol forhistone marks down to 10,000 cells.
• Provide accurate sequencing library preparationfrom just picogram inputs
Quality control analyses are expedited with theFragment Analyzer™ Automated CE System from AATI;
a fast, high resolution parallel capillary electrophoresis(CE) instrument that provides accurate sizing andquantification down to 1,000 cells. The routine quality
control checkpoints, after shearing of DNA and after
library preparation, can be completed with limited
number of cells on the Fragment Analyzer using theDNF-474 High Sensitivity NGS Fragment Analysis Kit,
replacing high concentration dependent agarose gelelectrophoresis. In this context, the Fragment Analyzer:• Conserves the ChIP’ed sample for sequencing withsuperior quantification sensitivity• Accurately sizes the sheared DNA smears through6,000 bp with only 1,000 cells• Automatically quantifies library samples• Improves overall ChIP-seq quality control analysis

Introduction:
With advances in next-generation sequencing (NGS), theprimary method for genome-wide mapping of proteinDNA interactions is chromatin immunoprecipitation withNGS detection (ChIP-seq) (Figure 1). ChIP-seq allows
for the detection and characterization of transcription
factor binding sites or patterns of histone modifications
for the entire genome.
Traditionally, ChIP-seq required high amounts ofstarting material, usually millions of cells, making rareor difficult cell types unsuitable for analysis. Diagenodehas developed groundbreaking solutions for epigenetic
studies on small sample sizes with the True MicroChIP
Kit and the MicroPlex Library Preparation™ Kit enabling
reliable analysis of protein-DNA interactions by ChIPPCR or ChIP-seq with inputs as low as 10,000 cells(1).
Conventionally, ChIP-seq quality control checkpoints
required agarose gel electrophoresis with a minimum
input of 50,000 cells for reliable quality assessment.
The Fragment Analyzer™ Automated CE System from

Method Overview:
Low Input Chromatin Shearing and Sizing
The first critical step of a successful ChIP experimentis the preparation of sheared chromatin with a suitable
fragment size range which is representative of thebiological scenario of interest. The Bioruptor® Pico from
Diagenode uses ultrasound ACT (Adaptive CavitationTechnology) in a temperature-controlled environment
to efficiently shear chromatin, providing high quality
chromatin with preserved epitopes for ChIP-seq.
Quality control checks prior to immunoprecipitationof the protein of interest are conventionally done withagarose gel electrophoresis, which requires a minimumof 50,000 cells for reliable visualization. This becomes aproblem when working with a limited population of cells.
Chromatin from HeLa cells were crosslinked and sheared
from 100 bp to 600 bp fragments using the Bioruptor®
Pico for small volume samples. After reversal of crosslinking and purification, samples were separated using
the Fragment Analyzer with the HS NGS Fragment Kit(Figure 2A&B) or agarose gel electrophoresis (Figure2C).

Figure 1- ChIP-seq workflow overview. Fragment Analyzer
replaces agarose gel electrophoresis at critical quality control
checkpoints.

Advanced Analytical Technologies, Inc. (AATI) offers quick
assessment of chromatin quality, size and quantification
with as little as 1,000 cells with the DNF-474 HighSensitivity NGS Fragment Kit (HS NGS Fragment Kit)(2,3).
The low sample input requirement makes it the perfect
replacement for agarose gel electrophoresis. Diagenode’s
ChIP solutions in combination with Fragment Analyzer
from AATI offer a complete solution to the ChIP-seq
high cell input dilemma.

The Fragment Analyzer offers highly accurate andsensitive analysis of chromatin shearing with as little
as 1,000 cells. PROSize® Data Analysis Software
automatically provided a smear size and concentration
while also offering a feature to overlay sample profiles foreasy visualization of the targeted DNA (Figure 2A). The
electropherogram overlay displayed that all the smears
had a similar shape regardless of the initial cell count.
This confirms consistent chromatin shearing among
samples from different cell counts with the Bioruptor®
Pico from Diagenode. Chromatin shearing by agarose
gel electrophoresis was best visualized with a sample
from 50,000 cells.
With confident analysis over a broad dynamic
concentration range, the Fragment Analyzer can be usedin place of agarose gel electrophoresis not only for small
1,000 cell sample size, but also for larger 50,000 cellsample sizes. Sheared chromatin quality control checks
with the Fragment Analyzer offers analysis over a wide
range of sample sizes, while reporting distribution profiles
and accurate sizing results important for successful
chromatin immunoprecipitation.

fast separation, automatic sizing, and quantification oflow concentration samples, confirming the quality of alibrary for sequencing.

C50,000

C

1,000
2,500
5,000
10,000B

20,000A

Figure 3- Quality control check of a ChIP-seq library on theFragment Analyzer. High Efficiency ChIP performed on
10,000 cells.
Figure 2- Chromatin analysis after purification. (A) With theFragment Analyzer. (B) Separation of 1,000 cells with theFragment Analyzer. (C) With agarose gel electrophoresis.

Reliable Chromatin Immunoprecipitation and Library
Preparation from 10,000 Cells and Pre-Sequencing Quality
Control Check
The True MicroChIP Kit allows performing a highefficiency ChIP on as few as 10,000 cells.
The successful immunoprecipitation obtained withH3K4me3, H3K27me3, H3K9me3, and H3K27me3
antibodies was confirmed by qPCR which shows goodenrichments in positive control regions and negligible
signal in negative control regions. In ChIP, checking forenrichment is crucial before proceeding with library
preparation. Please see https://www.diagenode.com/
img/documents/application-notes/AATI_Diagenode_qPCR.jpg to view the enrichment data from this assay.
NGS-ready libraries were constructed with 30 pg of
immunoprecipitated DNA using the MicroPlex Library
Preparation Kit. Thus, the True MicroChIP Kit used inconjunction with the MicroPlex Library Preparation™
Kit and premium grade antibodies enable ChIP-seq withjust a few picograms of input.
Prior to sequencing, it is necessary to check the qualityof the NGS library to ensure successful results. TheTM
Fragment Analyzer Automated CE System offers
quick analysis of the integrity, molarity, and size ofthe DNA smear before sequencing. After library
amplification, the samples were separated on theFragment Analyzer to confirm distribution profile andsizing results (Figure 3). The Fragment Analyzer offers

Bioinformatics Analysis
Bioinformatics analysis after sequencing displayed
excellent results from low amounts of starting material.
Comparison of 30 pg of DNA extracted from 10,000
cells and 300 pg of DNA extracted from a higher cellnumber was performed.
The 30 pg dataset generated low background noiseand highly reliable enrichments (peaks), which wereconfirmed by both the 300 pg dataset and theH3K4me3 dataset generated by the Broad Institute forthe ENCODE project (Figure 4). The 30 pg dataset
also fulfilled the criteria for the Top40 overlap ratio(1).
Bioinformatics analysis confirms that Diagenode andAATI have provided a successful pathway for small input
sample analysis with ChIP-seq.

Figure 4- Screenshot of the IGV Genome Browser comparing
the peaks of different datasets. The top line (green) is the30 pg dataset, the middle line (blue) shows the 300 pg
dataset, and the bottom line (red) is the Broad Institute data.
Both the 30 pg and 300 pg fulfill the Top40 overlap ratiocriteria.

Summary
Diagenode and AATI are the first to provide a reliable and comprehensive workflow for small sample analysis withChip-seq. A combination of Diagenode’s True MicroChIP Kit and the MicroPlex Library Preparation Kit and AATI’s
TMFragment Analyzer Automated CE System with the HS NGS Fragment Kit enables reliable detection of proteinDNA interactions by ChIP-seq with inputs as low as 10,000 cells and quality control checks from as little as 1,000
cells, thus advancing epigenetics research.
References	1. “From miniscule amounts to magnificent results: reliable ChIP-seq data from 10,000 cells with the True
MicroChIP™ and the MicroPlex Library Preparation™ kits”. Diagenode, Inc. www.diagenode.com/files/
application_notes/True_MicroChIP_and_MicroPlex_kits_Application_Note.pdf
2. “High Sensitivity NGS Fragment Analysis Kit”. Advanced Analytical Technologies, Inc. www.aati-us.com/
consumables/reagent-kits/fragment-analyzer/dnf-474/
Author Information
 ffiliations
A
Diagenode s.a., Liége, Belgium.
Wassim Lakhal, Irina Panteleeva, Jerome Kroonen, Celine Sabatel, Miklos Laczik & Agnieszka Zelisko-SchmidtDiagenode Inc., Denville, New Jersey, USA.
Rini Saxena
Advanced Analytical Technologies, Inc., Ankeny, Iowa, USA.
Benjamin Deist, Chava Pocernich & Steve Siembieda

Advanced AnalyticalTechnologies, Inc.
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USA
Phone: (515) 964-8500
E-mail: techdata@aati-us.com

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