I am working with the True MicroChIP & Microplex Library Preparation Kits and several histone modification antibodies like H3K27ac, H3K4me3, H3K36me3, and H3K27me3. I got always very good and reproducible results for my ChIP-seq experiments.
Andrea Thiesen, ZMB, Developmental Biology, Prof. Dr. Andrea Vortkamp´s lab, University Duisburg-Essen, Germany
about H3K27ac Antibody, H3K27ac Antibody (sample size), H3K4me3 Antibody, H3K4me3 polyclonal antibody (sample size), H3K36me3 Antibody, H3K36me3 Antibody (sample size), H3K27me3 Antibody, H3K27me3 antibody (sample size), True MicroChIP-seq Kit, MicroPlex Library Preparation Kit v2 (12 indexes).
Morten Skage and Dr Ave Tooming-Klunderud, Norwegian Sequencing Centre, Oslo, NorwayThe Norwegian sequencing centre (NSC) is a national core facility fully equipped to handle a diverse set of sequencing projects. We see an increased interest in long read technologies, in which our PacBio Single Molecule sequencing service is important. PacBio, (or SMRT) sequencing, like any other single molecule system, starts with a critical library preparation step to generate long fragment libraries of 10-20kb length. Even longer libraries can be made, but common to all library types is to start with good quality DNA of high molecular weight. Physical shearing of the DNA, targeting the desired library length, is the first critical step in the procedure. NSC has experience with different methods to fragment DNA like nebulization, enzymatic, tagmentation, covaris AFA, g-tubes (also covaris), custom syringe/needle protocols and hydroshearing. The best fragmentation method should produce a sharp fragmentation pattern close to the target length as defined by the protocol settings, with as little “smear like” pattern of lower molecular length as possible. The final step of PacBio library preparation is to size select only the longest fragments (> 7kb) as this increases the overall success rate of only sequencing the longest library fragments. It is not uncommon to loose a lot of material during this step. However, if the initial fragmentation yields sharp fragmentation patterns with minimal smear, more of the total DNA is retained in the final library. This is perhaps most important on samples were DNA amounts are limited. The optimal fragmentation method has to produce similar fragmentation pattern each time, with little deviation with sample type and input amount. We have found the new Megaruptor from Diagenode to be one of the best instruments for routine shearing of DNA to lengths of 2-40kb. It is easy to use, with walk away shearing, and the instrument does all the washes before, in between samples and after shearing without user intervention. Disposable hydropores is beneficial for lowering the contamination risk. The fact that it is so easy to use make it a good choice for laboratories with multiple users and/or when training new staff.
I am working with Diagenode's antibodies for over 5 years now. The great and specific performance as well as the reliable high quality of the antibodies are crucial for the success of my ChIP experiments.
Dr Holger Bierhoff, Division Molecular Biology of the Cell II, Prof. Dr. Ingrid Grummt’s lab, DKFZ, Heidelberg, Germany
To expedite the larger-scale phenotyping of pollen number difference within Arabidopsis family, we have modified an existing method more suitable for our purpose of pollen counting using a Bioruptor®. We grew four plants per genotype. Three flower buds per plant were harvested and dried at 65°C overnight. We collected flower buds with mature pollen, but before the anthers were opened (flower stage 13). We did not use the first and second flowers of the inflorescence, as they tend to show developmentally immature flower morphologies. 30 μl of 5% Tween-20 is added to the dried flower and samples are sonicated using the Bioruptor® Plus with high power mode for 10 cycles (sonication cycle: 30 sec ON, 30 sec OFF) to separate pollen grains from the anthers. Then, all pollen solutions are measured with a cell counter.
Misako Yamazaki, Evolutionary and Ecological Genomics (Kentaro Shimizu group), Institute of Evolutionary Biology and Environmental Studies, University of Zurich, Switzerland
about Bioruptor® Plus sonication device.
We are particularly satisfied with the Bioruptor® Pico that we have recently purchased. Since we have started using this novel machine, all experiments for our epignetics research have been much more easy to perform. This machine is really a great thing for us. The whole department has now started using this novel device.
Dr. Marco Pontoglio, Head of the Team "Expression Génique, Développement et Maladies" (EGDM) and Director of the “Development, Cancer and Reproduction”, Department Institut Cochin, Université Paris-Descartes
Diagenode has provided a Bioruptor® Pico demo instrument to us for 2 weeks in September. We were getting very reproducible results between different tubes in the same sonication as well as between replicate experiments. We have obtained considerably improved results especially for cross-linked chromatin from mammalian cells that has always been a problem for us with the old Bioruptor. A bonus also was a reduced sonication time, although the preparation time is slightly delayed due to the glutinous nature of the beads.
Dr. Tatyana Nesterova, Department of Biochemistry, University of Oxford
about 15 ml Bioruptor® Pico Tubes & sonication beads.
The new Bioruptor® Pico machine has reduced the amount of time spent sonicating Chromatin by a massive amount. Some protocols require quite harsh fixing conditions which meant fragmenting DNA on the old machine was taking many rounds and several times. With the new Bioruptor® Pico machine these sonications were taking just one round of 10 cycles thereby reducing the fragmentation time substantially. Following sonication, I have used the new IDeal ChIP-seq kit. This is a nice straight forward kit that if followed with an appropriate chip validated antibody gave amazing chip-seq results that worked time and again with several different transcription factors. I would recommend both kits for good, consistant chromatin work.
Dr. Karen Dawson, RNA Biology Group, Cancer Research UK Manchester Institute at the University of Manchester
about iDeal ChIP-seq kit for Histones, iDeal ChIP-seq kit for Transcription Factors.
We used the MicroPlex version 2 kit to generate libraries using ChIP DNA for several transcription factors and compared the results to a standard library generation protocol starting from 5ng of ChIP DNA. Even when we reduced the starting amount of DNA by 10-fold, the MicroPlex Kit produced the same high yields and quality of the libraries. As expected, the number of duplicate reads increased but 15 to 20 million unique reads were sufficient to achieve excellent enrichment data. We found that no information was lost, and the MicroPlex Kit helped produce data that was consistent with the standard protocol despite the lower input. On top of this, the MicroPlex Kit was extremely user-friendly and saved us time. The MicroPlex version 2 kit will make challenging ChIP-seq experiments that rely on very limited amount of starting material much easier with robust results.
Katia Basso, PhD, Assistant Professor, Columbia University, New York
about MicroPlex Library Preparation Kit v2 (12 indexes).
When performing FAIRE-PCR (Formaldehyde-Assisted-Isolated-Regulatory-Elements) experiments, instead of using ethanol precipitation after the phenol-chloroform extraction, we used the Ipure kit to decrosslink and purify DNA. The yield was much better than after ethanol precipitation. In addition, we get rid of problem of inhibition of the following PCR reactions.
Dr Emmanuèle Mouchel-Vielh, CNRS-UPMC Laboratoire de Biologie du Développement, Paris, France
The Diagenode Plant ChIP-seq kit works very well. The kit gives higher enrichment over background for the positive samples compared to our previous method. Using both the Plant ChIP-seq Kit and Diagenode’s Premium H3K9/14 ac polyclonal antibody, we performed ChIP-qPCR of H3K9/14 ac in region 3 of the TOC1 promoter. TOC1 is a circadian clock gene involved in evening loop that inhibits circadian clock genes expressed during the light phase of the day. We observed higher acetylation around TSS of TOC1 at the end of light phase in short day conditions, results that correlate with previously published data.
Dorota Komar, Centre for Plant Biotechnology and Genomics, Madrid, Spain
about H3K9/14ac Antibody, H3K9/14ac Antibody (sample size).
The Diagenode MicroPlex kit is an efficient way to make sequencing libraries, especially from samples with very low inputs. We used it for successfully processing ChIP material of transcription factors and histone modifications.
Dr. Filippo Cernilogar (Ludwig-Maximilian-Universität München, Germany)
When doing CRISPR experiments, it is essential to verify the expression levels of the Cas9 to ensure high-quality results. Diagenode's new CRISPR/Cas9 4G10 antibody is extremely specific, able to recognize different Cas9 fusion proteins (even those used for CRISPRi experiments) and compatible with many biochemical assays, such as immunofluorescence, western blot and immunoprecipitation. Among the different antibodies in the market, Diagenode's 4G10 CRISPR/Cas9 is our antibody of choice.
Researcher at EPFL, Lausanne, Switzerland
about CRISPR/Cas9 Antibody 4G10 (sample size), CRISPR/Cas9 Antibody 4G10, CRISPR/Cas9 Antibody 4G10.
The Diagenode MicroPlex kit is the quickest and most efficient way to make sequencing libraries, especially from samples with very low inputs. We regularly start with picogram amounts of ChIP material and produce excellent quality libraries that would be impossible to make using normal methods. Sequencing libraries made from the MicroPlex kit give us excellent results even in large genomes. The kit performs very well, and we will use the kit in the future for studies with low cell numbers or starting material.
Dr. Morgan Sammons, Lab of Dr. Shelley Berger, University of Pennsylvania
about MicroPlex Library Preparation Kit v2 (12 indexes).
Not only does the IP-Star eliminate the problem of human variation associated with producing our samples, it also enables us to produce 1000-2000 ChIP-seq samples per year very reliably. The IP-Star reduces our processing time down from one day of manual work to just one overnight run with only 30 minutes of hands-on work. The IP-Star has made all our ChIPs consistent and the process completely reliable regardless of the operator or the time of day
Dr. John Lambourne, Postdoctorate Researcher at the Innovation Centre, McGill University, Canada
We are very happy with the SX-8G IP-Star that we are routinely using in combination with MeDIP or MethylCap for DNA methylation profiling or for ChIP of histones or transcription factors. The use of the robot significantly reduces the hands-on time, and even more importantly provides a very high reproducibility, which is very valuable and essential for the generation of large number numbers of precious samples that will be analyzed on NGS platforms
Henk Stunnenberg, The Radboud University Nijmegen Medical Centre
We found that the IP-Star® provides an efficient, reliable and accurate tool for the construction of Illumina next-generation RNA-seq libraries, especially for trancriptome-based annotation of larger genomes or genomes with many alternative gene isoforms. The automated protocol significantly saves on-hands experimentation time, related costs and error-prone manual steps. Added benefits include ease of operation and generation of consistent data regardless of human variability and experimental run. Adaptation of this technology should support the unveiling of the mechanisms governing differential gene expression and transcription processing genome-wide, leading to a better understanding of genetic and epigenetic regulation and inheritance in a time-efficient manner.
UB Genomics and Bioinformatics Core, State University of New York at Buffalo, Research Team: Dr N. Nowak, Dr M.J. Buck, Dr M. Tsompana, S. Valiyaparambil, J. Bard, and B. Marzullo.
Automated ChIP experiments in the IP-Star® Compact Automated System can be used to detect changes in transcription factor binding after treatment with small interference RNAs. Compared to conventionnal ChIP, the use of the SX-8G IP-Star® Compact in combination with the Auto ChIP kit saves working time and improves the reproducibility of ChIP experiments
S. Prall, Northern Institute of Cancer Research, Newcastle University
I was using Diagenode Plant ChIP-seq kit to investigate abundance of particular histone mark in A.thaliana's seedlings. Plant ChIP-seq kit yielded in high amounts of immunoprecipitation DNA, very good relative enrichment to input and low background level. I frankly recommend it for ChIP on Plant tissues.
Msc Pawel Mikulski, Lab of Dr. Daniel Schubert, Heinrich Heine University Düsseldorf
The ability to produce libraries starting with low input DNA is primordial when using ChIP-seq. I am a ChIP-seq user and I always use the MicroPlex kit for library making from my immunoprecipitated DNA. Not only it’s quick and requires as little as 50 pg of input DNA but it also never fails. I’ve made over 100 libraries using the MicroPlex library preparation kit, and none of them failed. I use the Microplex kit for preparing libraries from immunoprecipitated DNA and from genomic DNA with low concentration.
Zineb Rchiad, Microbial Genomics Laboratory, King Abdullah University of Science and Technology, Kingdom of Saudi Arabia.
As part of a genomics lab, I regularly prepare libraries from a large number of samples. The IP-star® is extremely useful for high throughput library preparation. I have prepared 200 genomic libraries in less than 1 month using the IP-star®, it has helped me economize time and effort and increase reproducibility. I have used the IP-star® for automation of ChIP as well; 16 samples can be processed in one run. In brief, it’s a blessing to have this machine if you apply ChIP-seq to a large number of samples.
