PROTOCOL

Re-shearing of decrosslinked immunoprecipitated
DNA using the Bioruptor® PicoINTRODUCTION
ChIP-seq is a common tool used to study protein-DNA
interactions in cells and tissues on a genome-wide scale.
The size of the immunoprecipitated DNA fragments isa key parameter for successful library preparation andreliable ChIP-seq data. Re-shearing of the purified decrosslinked DNA after immunoprecipitation and before
library preparation enable enrichment of fragmentsin the desired optimal size range suitable for nextgeneration sequencing (Figure 1). This approacheliminates a size-selection step from a conventional
library prep workflow and increases the yield ofimmunoprecipitated DNA available for a library. It
improves the sensitivity and/or resolution of standard

ChIP-seq experiments, especially for inactive histonemarks (Laczik et al, 2016). The chromatin that represents
inactive marks is more resistant to sonication. As aconsequence, these regions are relatively more present
among the longer fragments after the initial shearing
step, and re-shearing shifts these fragments to smaller
sizes which are more efficiently incorporated intosequencing libraries. ChIP-seq on active histone marks,transcriptional factors or chromatin modifying proteins
benefits from the re-shearing approach by increasing
the number of fragments available for library generation
for sequencing and permitting reduction of the starting
amount of cells.

REQUIRED MATERIALS AND EQUIPMENT• Bioruptor® Pico (Diagenode, Cat. No. B01060001)• 0.2ml tube holder (Diagenode, Cat. No. B01200044) and corresponding 0.2 ml microtubes for
Bioruptor® Pico (Diagenode, Cat. No. C30010020) or 0.1 ml tubes holder (Diagenode, Cat. No. B01200041)
and corresponding 0.1 ml microtubes for Bioruptor® Pico (Diagenode, Cat. No. C30010015)• Library preparation Kit (e.g. MicroPlex Kit, Diagenode, Cat. No. C05010012 or iDeal Library Preparation Kit,
Diagenode, Cat. No. C05010020).
• (Optional) Fragment Analyzer™ (Advanced Analytical) or BioAnalyzer (Agilent) with high sensitivity reagents

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PROTOCOLStandard
methodRe-shearing
methodCell collection
Crosslinking
Lysis
Chromatin shearing

Cell collection
Crosslinking
Lysis
Chromatin shearing

ChIP & elution

ChIP & elution

Unspecific fragments
discarded

Unspecific fragments
discarded

Decrosslinking& purificationSize-selection

Fragments outsideof size range discarded

Library preparation
Sequencing

Decrosslinking& purification

Iterative reshearing

Library preparation
Sequencing

Figure 1. An overview of standard method (left) and re-shearing method (right)

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Europe Diagenode sa / LIEGE SCIENCE PARK // Rue du Bois Saint-Jean, 3 // 4102 Seraing (Ougrée) // Belgium // Phone: (+32) 4 364 20 50 // E-mail: info@diagenode.com
USA Diagenode Inc. / 400 Morris Avenue, Suite 101 // Denville, NJ 07834 // USA // Phone: (+1) 862 209-4680 // Mail: info.na@diagenode.com

PROTOCOLPROTOCOL
1. Perform chromatin immunoprecipitation using an appropriate protocol (e.g. Diagenode iDeal ChIP-seq
Kit for Histones (Cat. No. C01010051) or iDeal ChIP-seq Kit for Transcription Factors (Cat. No. C01010055)
and ChIP-seq grade antibodies (www.diagenode.com/categories/antibodies). Ensure that an expectedenrichment is achieved.
2. Transfer 20 µl of immunoprecipitated DNA to Bioruptor® microtubes (0.2 ml or 0.1 ml).
3. Vortex and spin down samples and proceed to sonication in the Bioruptor® Pico with the following settings:
 Sonication cycle: 30 sec ON/30 sec OFF
 Total sonication time: 5 -10 cycles
 Temperature: 4°C

Note: To guarantee homogeneity of sonication, the tube holder should be always completely filled with tubes.
4. (Optional) if DNA concentration is sufficient (at least 2-3 ng/µl), DNA fragment size distribution before
preparing libraries can be assessed on the Fragment Analyzer™ (Advanced Analytical) using the DNF-474
High Sensitivity NGS Fragment Analysis Kit or BioAnalyzer (Agilent) using High Sensitivity DNA Analysis kit.
5. Proceed to the library preparation using re-sheared DNA.

Note: A size selection step can be skipped.

Figure A

Figure B

Figure 2. The re-shearing method enriches fragments in an optimal size range suitable for next-generation sequencing.
Shearing profile of immunoprecipitated chromatin before (Figure A) and after (Figure B) re-shearing. Chromatin from HeLa cells was preparedaccording to the iDeal ChIP-seq Kit for Histones and sheared using the Bioruptor® Pico for 8 cycles. ChIP was performed with an H3K27me3
antibody (Diagenode, Cat. No. C15410195). De-crosslinked and purified DNA was analysed on a BioAnalyzer. The observed fragment range
is compatible with ChIP-seq library preparation.

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PROTOCOL
LIST OF DIAGENODE ANTIBODIES SUCCESSFULLY TESTED USINGTHE RE-SHEARING METHOD:ProductCat. No.

Product

Cat. No.

H3K27me3Cat. No. C15410195

NFkB

Cat. No. C15310255

H3K4me3Cat. No. C15410195

E2F6

Cat. No. C15410314

H3K4me1Cat. No. C15410194

RNF2

Cat. No. C15410313

p53

Cat. No. C15410083

OCT4

Cat. No. C15410305

H3K27me3S28p

Cat. No. C15410091

H3K36me3Cat. No. C15410058

H4K20me3Cat. No. C15410057

NFYB

Cat. No. C15410241

HDAC2

Cat. No. C15200201

KAP1

Cat. No. C15410236

EZH2

Cat. No. C15200180

H3K9me3Cat. No. C15410193

LSD1

Cat. No. C15410028

CBX8

Cat. No. C15410333

CTCF

Cat. No. C15410210

REFERENCES
Laczik et al. Iterative Fragmentation Improves the Detection of ChIP-seq Peaks for Inactive Histone Marks.
Bioinformatics and Biology Insights 2016:10 209–224 doi: 10.4137/BBI.S40628
Mokry M, Hatzis P, de Bruijn E, Koster J, Versteeg R, Schuijers J, et al. (2010) Efficient Double Fragmentation
ChIP-seq Provides Nucleotide Resolution Protein-DNA Binding Profiles. PLoS ONE 5(11): e15092. doi:10.1371/
journal.pone.0015092

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Europe Diagenode sa / LIEGE SCIENCE PARK // Rue du Bois Saint-Jean, 3 // 4102 Seraing (Ougrée) // Belgium // Phone: (+32) 4 364 20 50 // E-mail: info@diagenode.com
USA Diagenode Inc. / 400 Morris Avenue, Suite 101 // Denville, NJ 07834 // USA // Phone: (+1) 862 209-4680 // Mail: info.na@diagenode.com