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Optimized Protocol for Robust Chromatin Shearing andImmunoprecipitation of Human Pancreatic Islets using
the Covaris® Focused-ultrasonicatorAuthors:

Irene Miguel-Escalada1, Xavier Garcia1, and Jorge Ferrer1,2

Affiliation: 1 Genomic Programming of Beta-cells Laboratory, Institut d'Investigacions August PiiSunyer (IDIBAPS), 08036 Barcelona, Spain
2 Department of Medicine, Imperial College London, London W12 0NN, United Kingdom

Abstract

In this application note, we present an optimized protocol

Epigenomic profiling of human islet (HI) cells allows investigators

to process human pancreatic islets using the Covaris S220.

to understand the molecular mechanisms governing tissue-specificAcoustic shearing provides fragments usable for highly sensitive

regulatory functions and disease. Here, we developed a chromatin

downstream applications such as ChIP-seq and ChIP-qPCR.

shearing and immunoprecipitation (IP) protocol of fixed isolated
human pancreatic islets. Demonstrated here, we show how theComparison Between Covaris Focused-ultrasonicator andCovaris S220 Focused-ultrasonicator provides highly reproducible

Bioruptor and Probe Sonicators

chromatin shearing, resulting in improved signal-to-noise ratios

Compared to standard probe sonicators, where the metal probe

and sensitivity for ChIP-seq or ChIP-qPCR.

comes in direct contact with the human sample, chromatin
shearing using the Covaris S220 Focused-ultrasonicator provides aIntroduction

non-contact robust shearing protocol. Furthermore, unpredictable

Why are we Studying HI?

foaming induced by metal probe sonicators is avoided to minimize

Profiling human primary tissues is necessary to gain insight intosequencing bias.

the molecular mechanisms that control organ development anddisease. In the case of human pancreatic islets, the motivation is

Unlike traditional water bath sonicators, chromatin shearing

driven by the worldwide growing incidence of diabetes mellitus,

is isothermal. The external chiller controls the water bathwhich currently affects more than 400 million people globally (1).

temperature, which eliminates the need to frequently stop the run

As a result, Human Islet (HI) research is critical to improve ourdue to excessive heating, or to manually add ice chips to the tankunderstanding of its biology, however, the availability of pancreatic

between cycles.

islets from donors are very limited and considered a precious
The assessment of chromatin shearing patterns using the Agilent

resource to the research community.

High Sensitivity DNA chip revealed that this protocol allows for theWhat are the Challenges of Studying HI?

enrichment of fragments in the 200 to 700 bp range. In addition,

Traditional ChIP sample preparation protocols are optimized forthe protocol also reduces the amount of high molecular weight

use with cultured cell lines, which are easy to disperse into single

chromatin which is commonly found after sonicating compact HI.

cells and lyse. In contrast, processing primary tissue is more

More importantly, improved shearing provides a higher recovery oftechnically challenging and requires optimization using precious

immunoprecipitated chromatin as shown in the RT-qPCR results.

and limited human material. HI of Langerhans are small clusters ofendocrine cells composed of several cell types including: insulin-This protocol is easy to follow, provides reproducible chromatinsecreting β-cells, glucagon-secreting α-cells, and somatostatin-

shearing across valuable human samples, and preserves protein-secreting δ-cells. The size and purity of these clusters can vary

DNA interactions. We have applied this protocol to create

greatly between donors and isolation centers, which pose technical

ChIP-seq libraries and map the binding sites of several histone

challenges to their processing and downstream applications.

modification marks, transcription factors and co-factors.

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Materials and Methods

DNA Isolation and Assessment of Shearing Efficiency

Covaris Instruments and Consumables

In order to optimize and assess the size distribution after- S220 Focused-ultrasonicator (PN 500217)

chromatin shearing, the equivalent of at least 100,000 cells were

- microTUBE AFA Fiber Pre-Slit Snap-Cap 6 x 16 mm

de-crosslinked and analyzed using an Agilent High Sensitivity		 (PN 520045)

DNA chip. Briefly, 1.5 µl of RNase A was added to the sheared

Reagents

chromatin and incubated at 37 °C for 30 min. Then, 5 µl of- 16% Paraformaldehyde (Agar Scientific, R1026)

Proteinase K was added followed by de-crosslinking at 65 °C- 1.25 M Glycineovernight. Next, DNA was purified using phenol-chloroform- Protease inhibitor cocktail (Sigma-Aldrich, 4693132001)

and precipitated with sodium acetate and ethanol. Between 5- Proteinase K 20 mg/ml (Roche, #03 115 852 001)

and 10 ng of purified the DNA was used to assess fragment size

- RNAse A 10 mg/ml (Qiagen, #19101)

distribution.

Buffers and Solutions

ChIP

For buffers and solution recommendations, please contact Covaris

The ChIP protocol was adapted from 2 published protocols (2,3).

Application Support at applicationsupport@covaris.com.

Sheared chromatin was diluted with a ChIP Dilution Buffer. Then,
30 µl of pre-blocked magnetic beads were added to preclear

Human Islet Culture and Fixation

chromatin by rotation for 1h at 4 °C. Between 1 and 3 µg of

De-identified isolated human pancreatic islets were received

the antibody of interest was added to the sample and incubatedfrom several European Isolation Centers in accordance to local

overnight at 4 °C while rotating. 50 µl of magnetic beads wereand national regulations. HI were placed on medium upon arrival

added to the sample and rotated at 4 °C for 2h. Beads wereand cultured for 72h. Next, the samples were manually picked

subsequently washed at 4 °C using a Low Salt Wash Buffer, Highup under a stereomicroscope and fixed with 1% formaldehyde

Salt Wash Buffer, LiCl Wash Buffer, and TE. Beads were eluted in

at ambient temperature for 10 minutes. For all purposes, we

300 µL of DNA elution buffer at 65 °C for 40 min with agitation.

assumed that each islet contained approximately 1000 cells.

Beads were placed on a magnet and supernatant was transferred

Formaldehyde was quenched with 10 mM Glycine for 5 min at

to a fresh tube and de-crosslinked together with input sample

ambient temperature and cells pelleted at 4 °C for 10 minutes atfollowing DNA isolation protocol above.

500 xg. After washing the sample twice with PBS supplemented
with protease inhibitors, the samples were snap frozen and stored

ChIP-qPCR and ChIP-seq

at -80 °C until further usage.

RT-qPCR was used to evaluate specific enrichment in at least
two positive and two negative genomic regions where theChromatin Shearing

protein of interest is known to be bound. Once the conditions

Fixed human islets were thawed on ice and subsequently lysed

were optimized, immunoprecipitated DNA was quantified andusing ice-cold Lysis Buffer for 15 to 20 minutes while frequently

sequenced on the Illumina HiSeq 2500. High quality reads wereforcing the sample through a 1 ml syringe attached to a 25G tomapped to hg19 genome and clonal reads were removed.

30G needle with care. Lysed cells were pelleted for 5 minutes at

MACS2 (4) was used to determine regions of statistically

500 xg at 4 °C and re-suspended in 130 µL of Shearing Buffer.

significant enrichment over input. UCSC Browser was used toThe sample was sheared using the following settings: Duty Factor

visualize the data.

(DF): 2%, Peak Incident Power (PIP): 105W, Cycles per Bust (CPB):
200, treatment time: 16 minutes.
Sheared chromatin was centrifuged at full speed for 15 minutes at
4 °C to remove debris and insoluble chromatin. Supernatant wastransferred to a fresh low-binding tube to proceed with the ChIP
assay and 5% of the lysate was stored at -20 °C to be used as theinput sample.

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Results and Discussion

Human pancreatic islet samples were processed and fixed usingA.

different mechanical shearing methods available to date. AabCovaris
S220
Covaris S220

the same conditions and theirCovaris
chromatinS220
sheared using three

distribution of fragments between 200 to 1000 bp with minimum
amounts of high molecular weight DNA was considered optimal.
Chromatin shearing using the Covaris S220 provided a distributionof fragment sizes that are mostly enriched in the 200 to 600 bp
range, which is suitable for successful sequencing (Figure 1A and B).

abcB.

In our experience with HI, the presence of high molecular weightfragments is difficult to avoidCovaris
when using
fixation times that areS220
bcompatible with probe and water bath sonicators. However, thebath sonicatoramountchromatin when using the Covaris S220
d of under shearedWater

Covaris S220
CovarisS220
Covaris S220
Probesonicator

is considerably lower when compared to the relative proportion ofunder sheared chromatin obtained using a metal probe sonicator(Figure 1C) or water bath sonicator (Figure 1D).

A.

C.

Covaris S220

1,6Covaris S220
Probesonicator
SonicatorWaterProbebathsonicatordPercentage of input (%)

bcdMediator ChIP qPCR

2

Water Bath Sonicator

Water bath sonicator

1,20,80,40
positive-1

Percentage of input (%)

D.

Waterbath sonicator
Water Bath Sonicator

positive-3

negative-1

negative-2

H3K27ac ChIP qPCR

B.

dpositive-2

5Covaris S220

4Probe Sonicator

32
1

positive-1

positive-2

negative-1

negative-2

Figure 2. ChIP qPCR results using human islets and several commercially available
sonicators. Enrichment of Mediator binding (A) and H3K27ac mark (B) arerepresented as percentage of input in several genomic regions where the factors are
known to be binding (positive1-3) and 2 negative regions (negative1-2).
Figure 1. Chromatin shearing in human islets is greatly affected by fragmentation
technique. Representative electropherograms of human islets fixed for 10 min
at ambient temperature and sheared using Covaris S220 (A and B), a metal probesonicator (C) or a water bath sonicator (D). The X axis measures DNA fragment size inbase pairs. The Y axis labels fluorescent units.

In order to assess the quality of the sheared chromatin to be used
for efficient ChIP reactions, we proceeded to enrich a protein
complex that is loosely bound to the DNA (the co-factor Mediator).
To start, HI were fixed and split in two equal parts. One sample
was processed using the optimized protocol with Covaris S220

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while the other sample was processed using a water bath sonicator. qPCR was performed using the enriched DNA revealed a higher signal
to noise ratio in the reactions performed using the Covaris S220. No enrichment in positive regions was observed when using a regularwater bath sonicator and equal ChIP conditions suggesting that the sonication conditions were either inefficient, or led to the destruction ofepitope or their release from the DNA (Figure 2A). Similarly, superior results were obtained after processing a different human islet sample
using the Covaris S220 when compared to a standard probe sonicator using the H3K27ac antibody (Figure 2B).
Several Mediator and Cohesin ChIP-seq libraries produced following this protocol were sequenced. A representative screenshot of PDX1
locus is shown in Figure 3, which depicts a robust and reproducible signal between human donors and expected co-occupancy patterns
with other histone modification marks previously mapped.

50 Kb

30 -

H3K27ac 0 _30 -Mediator 0 _30 -Mediator 0 _Cohesin
Cohesin30 -0_
30 0_

Figure 3. UCSC Browser screenshot of several human islet ChIP-seq samples on PDX1 locus. A region of chromosome 13 shows specific enrichment of Mediator and Cohesinbinding co-factors. Each track represents a different human pancreatic sample donor. Please note reproducibility between donors assayed for the same protein. H3K27ac ChIPseq track is added for reference.

Conclusion

References

The main challenge of working with primary human tissue is its

1. Global report on diabetes.World Health Organization 2016. ISBN 978 92 4		

inherent variability. The variation may be from the origin of the1565257 (http://apps.who.int/iris/bitstream/10665/204871/1/		9789241565257_eng.pdf)

tissue, isolation center, or cause of death; this will invariably affectthe reproducibility of downstream applications such as ChIP-seq.

2. Pasquali L. et al. Pancreatic islet enhancer clusters enriched in type 2 diabetes		

This sample preparation protocol optimized with the Covaris S220
Focused-ultrasonicator provides consistent chromatin shearingrisk-associated variants.Nat Genet. 2014;46(2):136-43.

3. Kagey MH. et al. Mediator and cohesin connect gene expression and chromatin

which is suitable for ChIP applications.		

Acknowledgments		

architecture.Nature. 2010;467(7314):430-5.

4. Zhang, Y. et al. Model-based analysis of ChIP-seq (MACS). Genome Biol.
2008;9(9):R137.

Irene Miguel-Escalada has received funding from the European
Union’s Horizon 2020 research and innovation programme under
the Marie Sklodowska-Curie grant agreement No 658145.

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M020070_RevB_Feb2019 | Information subject to change without notice | For research only | Not for use in diagnostic procedures | 2019© Covaris, Inc.

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