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DIAGENODE
BIORUPTOR
®
PLUS
USER MANUAL
Diagenode Inc. North America
/ Phone: +1 862 209-4680 // Fax: +1 862 209-4681 // Mail: orders.na
@
diagenode.com
resulting in broader size distribution or larger peaks. Bath water should be pure distilled water, changed
regularly.
2.
Sonication bath maintenance:
The sonication bath metal surface is fragile and requires a careful maintenance.
Use only soft sponge and distilled water to remove traces. Never use scratch scrub sponge since this would
alter the ultrasonic wave emitter surface.
Supplementary Data
Please note that there are three main sources of variation in both peak base-pair size and distribution:
1) The physical process of DNA fragmentation might not be entirely random in AT- or GC- rich regions.
2) The analytical process to determine fragment size has inherent variances (for example, gel electrophoresis and
microfluidics-based platform). Therefore, fragment distributions and peak values, even from technical replicates,
may not appear identical. If the sheared DNA sample will be resin or column purified or concentrated prior to analysis,
please remember to take out an aliquot for use as control prior to that step. Column purification and concentration of
the sheared DNA will generate a biased fragment distribution profile due to the inherent greater loss of the smaller
DNA fragments.
3) RNA contamination in genomic DNA preparation should be carefully removed using RNase-DNase free enzymatic
digestion since they might generate a biased fragment distribution profile on microfluidics-based platform (eg.
Agilent Bioanalyzer) or alter sonication effiency.
Chromatin shearing
Critical points for chromatin shearing
• Chromatin shearing efficiency varies on cell type.
Each cell type might need additional protocol optimization.
• The extent of cross-linking is critical for the efficient disruption of fixed cells and also affects DNA yield and average
size of chromatin fragments. Over-cross-linked chromatin will not produce small fragments, even by prolonged
sonication. Fix cells for 8-10 min at RT, always stop the reaction by glycine and wash 2-3 times with ice cold PBS.
• Cell density affects the sonication efficiency. Do not use too dense cell suspension. Optimal density is about
1-3x10^6/100 μl of sonication buffer.
• SDS is a key component of sonication buffer for chromatin shearing. Include 0.7-1% of SDS in your sonication buffer.
• Fresh formaldehyde for fixation.
Shearing of chromatin from adherent cell lines
For the adherent cells, we recommend to first harvest cells by trypsinization and perform chromatin cross-linking in
a cell suspension rather than on dishes as it results in a better reproducibility and consistency between experiments.
1. Discard medium to remove dead cells and wash cells by adding cold PBS.
2. Harvest cells by trypsinization.
3. Transfer cells in a tube containing 10 ml PBS (RT) and centrifuge 5 minutes at 1.300 rpm. Keep the cell pellet and
discard the supernatant. Wash the cells again in PBS.
Note:
At this step, cells might be counted.
4. Add PBS to a final volume of 500 μl for a
maximum of 10x10^6 cells
(for more cells, perform the fixation in a separate
tube).
5. Add formaldehyde to a final concentration of 1%, mix gently and incubate for 8-10 min at RT with rotation.
6. Stop the cross-linking reaction by adding glycine to a final concentration 0.125 M and incubate for 5 min at RT with
