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Summary of Contents for diagenode Bioruptor Standard
- Page 1 The Ultimate Guide for Chromatin Shearing Optimization with Bioruptor Standard and Plus ® USER'S GUIDE...
- Page 2 Not intended for any animal or human therapeutic or diagnostic use. © 2012 Diagenode SA. All rights reserved. No part of this publication may be reproduced, transmitted, transcribed, stored in retrieval systems, or translated into any language or computer language, in any form or by any means: electronic, mechanical, magnetic, optical, chemical, manual, or otherwise, without prior written permission from Diagenode SA (hereinafter, "Diagenode”) .
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Page 3: Table Of Contents
Content 1. Protocols and Guidelines 4-21 Critical points for chromatin shearing Tube holders & tubes for Bioruptor chromatin shearing ® Important additional comments about chromatin shearing Standard protocol for chromatin shearing from Chromatin Shearing Optimization kit-Low SDS A. Cell collection and DNA-protein cross-linking B. -
Page 4: Protocols And Guidelines
Protocols and Guidelines Critical points for chromatin shearing! • Chromatin shearing should be tested and optimized when one is starting a new ChIP project. • Given that every cell type may behave differently, it is highly recommended to optimize fixation and sonication conditions for each cell type before processing large quantities of cells or samples! One should perform an initial sonication time course experiment;... - Page 5 HeLa cells are fixed with 1% formaldehyde (for 8 min at room temperature (RT)). 100,000 cells are resuspended in 130 µl of buffer B from Diagenode’s Chromatin Shearing Optimization kit - High SDS (Cat. No. AA-003-0100) prior to chromatin shearing. Samples are sheared...
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Page 6: Important Additional Comments About Chromatin Shearing
Diagenode’s ChIP protocols o to ensure the best results, Diagenode strongly recommends to use fresh cells (compared to frozen cells) (Figure Note: In case of longer sonication time, it may also be helpful to assess the integrity of your epitope during the shearing process. - Page 7 Protocols and Guidelines Figure 3: Time course sonication experiment with the Bioruptor ® PLUS using the buffers and protocol from Diagenode’s Chromatin Shearing Optimization kit - Medium SDS HeLa cells are fixed with 1% formaldehyde (for 8 min at RT).
- Page 8 Figure 5: Superior chromatin shearing results obtained with fresh compared to frozen cells HeLa cells are fixed with 1% formaldehyde (for 8 min at RT). Nuclei isolation of 3x10e6 cells are performed using buffers from Diagenode’s chromatin Shearing Optimization kit - Low SDS (Cat. No. AA-001-0100) and are then resuspended in 100 µl of Shearing Buffer iS1 prior to chromatin shearing.
- Page 9 HeLa cells are fixed with 1% formaldehyde (for 8 min at RT). Nuclei isolation of 5 million cells are performed using buffers from Diagenode’s Chromatin Shearing Optimization kit - Medium SDS (Cat. No. AA-002-0100) and are then resuspended in 200 µl of Shearing Buffer S1 prior to chromatin shearing.
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Page 10: Standard Protocol For Chromatin Shearing From Chromatin Shearing Optimization Kit-Low Sds
Standard protocol for chromatin shearing with Shearing Buffers from Chromatin Shearing Optimization kit-Low SDS This protocol is based on Diagenode’s Chromatin Shearing Optimization kit - Low SDS (Cat. No. AA-001-0100) manual. Check the corresponding manual for more information (e.g. about buffer types and names). - Page 11 Protocols and Guidelines The protocol below is intended for adherent cells. Collect suspension cells by centrifugation and continue with the protocol starting from step 6. Pre-warm PBS, culture medium and trypsin-EDTA at 37°C. Remove the medium and rinse the cells with pre-warmed PBS (10 ml for a 75 cm culture flask).
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Page 12: Cell Lysis And Chromatin Shearing
Important: Do not freeze/thaw. 21. Proceed to Sheared chromatin analysis. C. Sheared chromatin analysis This protocol refers to Diagenode’s Elution Module (Cat. No. mc-magme-002) that can be ordered separately. Reagents not supplied: • RNase cocktail (e.g. Ambion, AM 2286 A) •... - Page 13 HeLa cells are fixed with 1% formaldehyde (for 8 min at RT). Nuclei isolation of 7.5 million cells are performed using buffers from Diagenode’s Chromatin Shearing Optimization kit - Low SDS (Cat. No. AA-003-0100) and are then resuspended in 500 µl prior to chromatin shearing.
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Page 14: Standard Protocol For Chromatin Shearing From Chromatin Shearing Optimization Kit-Medium Sds
Standard protocol for chromatin shearing with Shearing Buffers from Chromatin Shearing Optimization kit - Medium SDS This protocol is based on Diagenode’s Chromatin Shearing Optimization kit - Medium SDS (Cat. No. AA-002-0100) manual. Check the corresponding manual for more information (e.g. about buffer types and names). -
Page 15: Cell Lysis And Chromatin Shearing
Protocols and Guidelines • These are your cross-linked cells ready for chromatin shearing! • Do not disturb the pellet. NOTE: The fixed cells can be stored at -80°C for up to 4 months. However, we strongly recommend using freshly fixed cells for preparation of sheared chromatin prior to ChIP for ChIP-sequencing. -
Page 16: Sheared Chromatin Analysis
Important: Do not freeze/thaw. 30. Proceed to Sheared chromatin analysis. C. Sheared chromatin analysis This protocol refers to Diagenode’s Elution Module (Cat. No. mc-magme-002) that can be ordered separately. Reagents not supplied: • RNase cocktail (e.g. Ambion, AM 2286 A) •... - Page 17 46. Run samples (20 µl of DNA + 4 µl of 6x loading dye) on a 1.5% agarose gel. As an alternative run 1 µl on a microfluidic chip (e.g. Bioanalyzer High Sensitivity DNA chip). Figure 9: Agarose gel analysis of chromatin sheared with the Bioruptor PLUS using the buffers and protocol from Diagenode’s Chromatin ® Shearing Optimization kit - Medium SDS HeLa cells are fixed with 1% formaldehyde (for 8 min at RT).
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Page 18: Standard Protocol For Chromatin Shearing From Chromatin Shearing Optimization Kit-High Sds
Standard protocol for chromatin shearing with Shearing Buffers from the Chromatin Shearing Optimization kit - High SDS This protocol is based on Diagenode’s Chromatin Shearing Optimization kit - High SDS (Cat. No. AA-003-0100) manual. Check the corresponding manual for more information (e.g. about buffer types and names). -
Page 19: Cell Lysis And Chromatin Shearing
Protocols and Guidelines Add 13.5 µl of 36.5% fresh formaldehyde per 500 µl of sample (final concentration should be ~1%). NOTE: It is highly recommended to always use fresh formaldehyde Mix by gentle vortexing. Incubate for 8 min at RT to enable fixation. Optimization of fixation time may be required depending on cell type;... -
Page 20: Sheared Chromatin Analysis
Important: Do not freeze/thaw. 16. Proceed to Sheared chromatin analysis. C. Sheared chromatin analysis This protocol refers to Diagenode’s Elution Module (Cat. No. mc-magme-002) that can be ordered separately. Reagents not supplied: • RNase cocktail (e.g. Ambion, AM 2286 A) •... - Page 21 MCF7 cells (human breast adenomacarcinoma cells) are fixed with 1% formaldehyde (for 8 min at RT). From 1 million to 100,000 cells are resuspended in 130 µl of Buffer B from the Diagenode’s Chromatin Shearing Optimization kit - High SDS (Cat. No. AA-003-0100) prior to chromatin shearing.
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Page 22: Faqs
FAQs Frequenytly Asked Questions about chromatin shearing optimization with Bioruptor ® Fixation Cell lysis What is the recommended formaldehyde concentration? How can I achieve complete cell disruption? 1% formaldehyde in PBS (pay attention to always use Do not use too many cells in the cell lysis buffer. fresh formaldehyde). -
Page 23: Checking For High-Quality Shearing On Agarose Gel And Microfluidic Chip
What do smears indicate? range for your specific ChIP-seq application, please Gel electrophoresis of cross-linked samples often give a contact us: techsupport@diagenode.com. smear on the gel. Also, take several pictures of the gel to assure image quality. To obtain clearer image with accurate fragment size, reversion of the cross-linking (decross-linking) is strongly advised. - Page 24 400 Morris Avenue, Suite #101 Denville, NJ 07834 - USA Tel: +1 862 209-4680 Fax: +1 862 209-4681 orders.na@diagenode.com info.na@diagenode.com For a complete listing of Diagenode’s international distributors visit: www.diagenode.com/en/company/distributors.php For rest of the world, please contact Diagenode s.a. PR-Guide-CSh-V1-15_05_12 www.diagenode.com...
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