The Ultimate Guide for Chromatin Shearing Optimization with Bioruptor® Standard and Plus USER'S GUIDE FOR RESEARCH USE ONLY. 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”) . The information in this guide is subject to change without notice. Diagenode and/or its affiliates reserve the right to change products and services at any time to incorporate the latest technological developments. Although this guide has been prepared with every precaution to ensure accuracy, Diagenode and/or its affiliates assume no liability for any errors or omissions, nor for any damages resulting from the application or use of this information. Diagenode welcomes customer input on corrections and suggestions for improvement. TRADEMARKS The trademarks mentioned herein are the property of Diagenode or their respective owners. Bioruptor® is a registered trademark of Diagenode SA. Bioanalyzer is a trademark of Agilent Technologies. Gel Logic 1500 Imaging System & Kodak Molecular Imaging Software is a trademark of Kodak. 2 Content 1. Protocols and Guidelines 4-21 Critical points for chromatin shearing 4 Tube holders & tubes for Bioruptor® chromatin shearing 4 Important additional comments about chromatin shearing 6 Standard protocol for chromatin shearing from Chromatin Shearing Optimization kit-Low SDS 10 A. Cell collection and DNA-protein cross-linking 10 B. Cell lysis and chromatin shearing 11 C. Sheared chromatin analysis 12 Standard protocol for chromatin shearing from Chromatin Shearing Optimization kit-Medium SDS 1 4 A. Cell collection and DNA-protein cross-linking 14 B. Cell lysis and chromatin shearing 15 C. Sheared chromatin analysis 16 Standard protocol for chromatin shearing from Chromatin Shearing Optimization kit-High SDS 18 A. Cell collection and DNA-protein cross-linking 18 B. Cell lysis and chromatin shearing 19 C. Sheared chromatin analysis 20 2. FAQs 22-23 Fixation 22 Cell lysis 22 Number of cells/shearing buffer volume 22 Shearing buffer 22 Shearing conditions 22 Checking for high-quality shearing on agarose gel and microfluidic chip 23 3 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; evalution of the tests may be required to determine optimal shearing conditions. • Sample viscosity may have a major impact on sonication results. Careful homogenization of chromatin sample is strongly recommended before and during sonication processing. For optimal results, samples should be gently vortexed before and after performing 5 or 10 sonication cycles (depending on the protocol), followed by a short centrifugation at 4°C to recover sample volume at the bottom of the tube. When working with large cell numbers careful pipetting can help ensure better homogenization but it will not replace the vortexing step mentioned before. • Always use the highest available power setting (e.g. H: High Power on the Bioruptor® Plus) for all your chromatin shearing experiments. • After shearing, it is essential to produce chromatin fragments of a size suitable for ChIP and subsequent analysis of the immunoprecipitated DNA. Although chromatin fragments from 100 - 1000 bp are recommended for ChIP PCR or ChIP qPCR assays, the optimal size range of chromatin for ChIP-seq analysis should be between 100 and 600 bp. Larger chromatin fragments can negatively influence ChIP-seq data quality. Tube holders & tubes for Bioruptor® chromatin shearing For chromatin shearing, depending on the final shearing volume, we recommend the following tube holders: • From 100 – 300 µl: 1.5 ml tube holder for Bioruptor® Standard & Bioruptor® Plus (Cat. No. UCD-pack 1.5) with 1.5 ml TPX microtubes for Chromatin shearing (Cat. No. M-50001) (Figure 1). • From 50 – 100 µl: 0.5/0.65 ml tube holder for Bioruptor® Standard, Bioruptor® Plus and the corresponding shearing tubes (Cat. No. WA-005-0500) (Figure 2). To use the tube holder, remove the lower part by turning counterclockwise. Then place microtubes in the unit. Attach the lower part to the upper part of the adaptor. To guarantee homogeneity of chromatin shearing, the tube holders should always be completely filled with tubes. Never leave empty spaces in the tube holder. Fill the empty spaces with tubes containing the same volume of distilled water. 4 Protocols and Guidelines Figure 1: Superior chromatin shearing results obtained with Diagenode’s TPX 1.5 ml tubes compared to standard 1.5 ml tubes 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 during 1, 2, 3, or 4 rounds of 5 cycles of 30 sec ON/30 sec OFF with the Bioruptor® PLUS combined with the Bioruptor® Water cooler (Cat. No. BioAcc-cool) & Single Cycle Valve (Cat. No. VB-100-0001) at HIGH power setting (position H) using 1.5 ml TPX microtubes (Cat. No. M-50001) or 1.5 ml standard tubes (Brand B). The sheared chromatin is decross-linked prior to agarose gel electrophoresis. 10 µl of DNA are analyzed on a 1.5% agarose gel. The left and right lanes show the 100 bp DNA molecular weight marker (MW). As shown on the left part of the image, the optimal shearing conditions correspond to 3 rounds of 5 cycles (30 sec ON/30 sec OFF; means 1 min ON & OFF time) (fragments between 100 and 600 bp) using TPX 1.5 ml tubes. Figure 2: Efficient chromatin shearing with the Bioruptor® PLUS using small sonication volumes (50 µl) HeLa cells are fixed with 1% formaldehyde (for 8 min at RT). 500,000 cells (lanes 1, 2, and 3) are resuspended in 50 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 for several rounds of sonication cycles of 30 sec ON/30 sec OFF with the Bioruptor® PLUS combined with the Bioruptor® Water cooler (Cat. No. BioAcc-cool) & Single Cycle Valve (Cat. No. VB-100-0001) at HIGH power setting (position H) using shearing tubes (Cat. No. WA-005-0500). Between each sonication round, the samples are vortexed gently and then shortly centrifuged at 4°C to recover sample volume at the bottom of the tube. The sheared chromatin is decross-linked prior to agarose gel electrophoresis. 10 µl of DNA (equivalent to 500 ng as measured with Qubit) are analyzed on a 1.5% agarose gel. Left panel: The left lane shows the 100 bp DNA molecular weight marker (MW). Lane 1: 2x 5 cycles (each 30 sec ON/30 sec OFF); Lane 2: 3x 5 cycles (each of 30 sec ON/30 sec OFF); Lane 3: 4x 5 cycles (each 30 sec ON/30 sec OFF). Right panel: Gel image was captured with Gel Logic 1500 Imaging System (KODAK). Lane 1 was scanned using Kodak software (V.4.0.5) to estimate DNA size distribution. Using shearing volume as low as 50 µl, optimal DNA size range for ChIP and ChIP-seq analysis can be easily obtained using Bioruptor® shearing tubes (Cat. No. WA-005-0500) (see lane 3). 5 Protocols and Guidelines Important additional comments about chromatin shearing 1. Cross-linking duration • 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 and should be avoided. • Fix cells for 8 min (default time) and up to 10 min at room temperature (RT), always stop the reaction by glycine and perform thorough washes to remove all traces of formaldehyde. Over fixation may have numerous negative effects on ChIP and/or ChIP-seq experiments and should be carefully monitored. First, longer fixation times (e.g. 10 - 30 min) may cause cells to form aggregates that do not sonicate efficiently. Second, for some antibodies, longer cross-linking time may increase background by increasing non-specific binding of epitopes to antibody. Finally, over fixation has been shown to generate artefacts such as sonication resistant 1.5 - 1.7 kb fragments. 2. Sonication time • Extensive sonication may alter antigenic properties and lead to loss of antigenecity during the immunoprecipitation step. Ideally, the least time course conditions that gives satisfactory results should be used (Figure 3). • For optimal fragmentation assessment, a serial dilution of sheared chromatin from 100 ng to 500 ng should be run on a 1.2 - 1.5% agarose gel (ideally the gel should be pre-stained). If only one unique sample has to be tested, we strongly recommend using de-cross-linked chromatin DNA to avoid any misleading size assessment using agarose gel electrophoresis (e.g. from 400,000 - 500,000 cells) (Figure 4). 3. Impact of cells on chromatin shearing • It is best to prepare samples of sheared chromatin from fresh cells and directly use them in ChIP, especially when doing ChIP-seq. However, it is also possible to freeze cells and shear chromatin another day. Pellets of frozen cells are by far more stable than sheared frozen chromatin samples (fresh cells > frozen cells >> frozen chromatin).