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DIAGENODE
BIORUPTOR
®
PLUS
USER MANUAL
Europe Diagenode sa
/ CHU - Tour GIGA - B34 - 3e étage // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) 4 364 20 50 // Mail: info
@
diagenode.com
rotation.
7. Wash cells 3 times with cold PBS
8. Resuspend cells in an appropriate volume of a Lysis buffer containing SDS (0.7-1%). 1x10^6 -3x10^6 cells/300 μl are
recommended for shearing in 1.5 ml tubes. Lyse cells on ice for 5-10 min. Vortex and centrifuge tubes before putting
in Bioruptor
®
.
Note
: Nuclei isolation is recommended when working with 3x10^6 cells to 10x10^6 cells. (Shearing ChIP kit from
Diagenode is available for this purpose, kch-redmod-100). Diagenode 1.5 ml TPX microtubes are recommended for
efficient chromatin shearing (Cat. No. M-50050 or M-50001).
9. Sonicate samples with Bioruptor
®
Plus with refrigerated sonication bath (or crashed ice sonication bath) for 10-20-30
cycles of 30 sec ON and 30 sec OFF at HIGH setting. Briefly vortex and centrifuge tubes after each run of 10 cycles.
10. Centrifuge samples at 14000 rpm for 5 min at 4°C and transfer the supernatant into a new tube. Use an aliquot of
sheared chromatin (equivalent of 100.000-500.000 cells) for analysis of shearing: perform a reversal of cross-links
and analyze on agarose gel. The remaining chromatin might be kept at -80°C.
Shearing of chromatin from suspension cell lines
Note
: Cells growing in suspension culture are known to be difficult to shear. Nuclei extraction is recommended before
sonication.
Do not use very dense cell suspension for sonication.
1. Cross-link chromatin with 1% fresh formaldehyde for 8-10 min at RT
2. Stop the cross-linking reaction by adding glycine to the final concentration 0.125 M for 5 min at RT with gentle
rotation.
3. Wash cells 3 times with cold PBS.
4. Extract cell nuclei and use isolated nuclei for shearing (Shearing ChIP kit from Diagenode is available for this purpose,
kch-redmod-100).
5. Resuspend nuclei in an appropriate volume of Lysis buffer containing SDS (1%). 1x10^6 – 3x10^6 cells/300 μl are
recommended for shearing in 1.5 ml tubes. Lyse nuclei on ice for 5-10 min. Vortex and spin down tubes before putting
in Bioruptor
®
.
Note:
Diagenode 1.5 ml TPX microtubes are recommended for efficient chromatin shearing (Cat. No. M-50050 or M-5001).
6. Sonicate samples with Bioruptor
®
Plus with refrigerated sonication bath (or crashed ice sonication bath) for 10-20-30
cycles of 30 sec ON and 30 sec OFF at HIGH setting. Briefly vortex and spin down tubes after each run of 10 cycles.
7. Centrifuge samples at 14000 rpm for 5 min at 4°C and transfer the supernatant into a new tube. Centrifuge samples
at 14000 rpm for 5 min at 4°C and transfer the supernatant into a new tube. Use an aliquot of sheared chromatin
(equivalent of 100.000-500.000 cells) for analysis of shearing: perform a reversal of cross-links and analyze in agarose
gel. The remaining chromatin can be kept at -80°C.
