We were very happy with the method. It gave good results in the end, and required much smaller samples than we need to reliably perform conventional ChIP-seq.
In our view, the main advantages of the ChIPmentation kit compared to our conventional ChIP-seq protocol are (most important first):
- smaller sample requirement,
- simpler workflow with less that can go wrong,
- slightly higher resolution and signal: noise ratio.Researcher from University of Nice-Sophia Antipolis, Nice, France
ChIPmentation sequencing profiles for p65. Chromatin preparation and immunoprecipitation have been performed on stimulated NIH3T3 cells using the iDeal ChIP-seq kit for TFs (Cat. No. C01010055). Chromatin from 4,000,000 cells was used for the immunoprecipitation in combination with either anti-p65 antibody or IgG. The library preparation was performed with the TAG Kit for ChIPmentation (Cat. No. C01011030) and 24 SI for ChIPmentation (Cat. No. C01011031).
about iDeal ChIP-seq kit for Transcription Factors, iDeal ChIP-seq kit for Transcription Factors, iDeal ChIP-seq Kit for Transcription Factors, 24 SI for ChIPmentation, TAG Kit for ChIPmentation.
One of our issues was that we could obtain only a limited number of cells, which is not enough for canonical ChIP-seq protocols. To solve this issue, we used the Diagenode ChIPmentation solution composed of iDeal ChIP-seq Kit for Transcription Factor, TAG Kit for ChIPmentation, and 24 SI for ChIPmentation. We performed ChIPmentation with IP-Star automated system for GATA6 in 2 million GATA6-overxpressing iPS cells. The result showed clear signal/noise ratio and was highly reproducible. This solution also worked in vitro differentiated definitive endoderm cells (data not shown).
Region 2Takahiro Suzuki, Ph.D., Senior Research Scientist, Cellular Function Conversion Technology Team, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan
Figure 1. ChIPmentation sequencing profiles for Gata6
Chromatin preparation and immunoprecipitation have been performed on hiPSCs (human induced Pluripotent Stem Cells) overexpressing Gata6 using the iDeal ChIP-seq kit for TFs (Cat. No. C01010055). Chromatin from 2,000,000 cells was used for the immunoprecipitation in combination with either anti-GATA6 antibody. The library preparation was performed with the TAG Kit for ChIPmentation (Cat. No. C01011030) and 24 SI for ChIPmentation (Cat. No. C01011031).
about iDeal ChIP-seq kit for Transcription Factors, iDeal ChIP-seq kit for Transcription Factors, iDeal ChIP-seq Kit for Transcription Factors, TAG Kit for ChIPmentation, 24 SI for Tagmented libraries.
We at PreOmics are using the Bioruptor® Pico on a daily basis along with the iST kits for our protein sample processing workflows. The ultrasonication-based lysis greatly improves cell and tissue disruption, as well as shearing of DNA, generating samples with significantly increased peptide and protein identifications. The Bioruptor Pico in our lab has successfully processed several thousands of samples such as cells, mammalian and plant tissues, bacteria or yeast. We are highly satisfied with the handling and reproducibility of the instrument and recommend it to improve sample preparation for protein analysis.Dr. Fabian Hosp, Head of Applications, PreOmics, Martinsried, Germany
about Bioruptor® Pico sonication device, iST cartridge holder for the Picoruptor (PreOmics).
As a PacBio Certified Service Provider it is critical that sample processing in my laboratory is precise and reproducible. For genome sequencing projects, the fragmentation of genomic DNA to precise and reproducible sizes is essential in order to optimize conditions for library preparation, sequencing, and downstream assembly. For this my laboratory relies on the Megaruptor system. The Megaruptor is the optimal system for long DNA fragment generation and tight fragment length distribution.Brewster Kingham, Delaware Biotechnology Institute, University of Delaware
about Megaruptor® 3.
The NGS Competence Center Tübingen (NCCT), together with four other national centers, has been established by the DFG (German Research Foundation) to support research projects with diverse needs of high-throughput sequencing technologies.
Long-read sequencing is very helpful to answer scientific questions in various topics such as microbiology or clinical research. We have noticed that the data yield of Nanopore sequencing can be notably increased by shearing the high molecular weight genomic DNA with an average size distribution of ~30kb and obtaining a read length N50 of 30kb. In this context, the Megaruptor 3 was critical to achieve long, homogenous and reproducible DNA preparation.
Megaruptor 3 is able to shear different molecular weight ranges up to 100kb; provided the input genomic DNA is of high-molecular weight. We have tested the Megaruptor 3 with genomic DNA from human blood, fibroblasts and difficult samples such as bacterial genomic DNA with high viscosity. With the Megaruptor 3 we have easily sheared up to 8 samples in parallel, saving preparation time. We have tested concentrations as low as 5 ng/µL and up to 70 ng/µL, saving sample material for optimization and meeting downstream requirements for library preparation.
Finally, handling of the Megaruptor 3 is quick, with a simple interface. Diagenode is fast in delivering consumables and these are ready-to-go. Sample preparation requires one pipetting step. You need to enter 2 parameters of your sample: volume and concentration in addition to the speed related to your desired size. It is a safe process without sample cross-contamination. It is easy to control whether the sample is going through the hydropore. The system is fast; for the concentration and speed conditions we have tested, runs were completed between half an hour and 2 hours.Elena Buena Atienza and Dr. Nicolas Casadei, Institute of Medical Genetics and Applied Genomics, University Clinics Tübingen
about Megaruptor® 3.
I have used Diagenode's antibodies for my ChIP–qPCR experiments in HK-2 cells. The antibodies performed very well in our experiments with specific signal, and good signal to noise ratio in the promoter of the gapdh gene.
Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K4me3 ChIP assays were performed using human HK-2 cells, the Diagenode antibody against H3K4me3 (Cat. No. C15410003) and optimized PCR primer pairs for qPCR. ChIP was performed with standard fixation and sonication protocol, using sheared chromatin from 4 million cells. A total amount of 4 ug of antibody were used per ChIP experiment, normal mouse IgG (4 μg/IP) was used as a negative IP control. Quantitative PCR was performed with primers specific for the promoter of the active genes GAPDH and. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). These results are in accordance with the observation that trimethylation of K4 at histone H3 is associated with the promoters of active genes.Claudio Cappelli PhD. Post-Doc. Investigator, Laboratory of Molecular Pathology, Institute of Biochemestry and Microbiology, University Austral of Chile, about H3K4me3 polyclonal antibody Premium, 50 μl size.
about H3K4me3 Antibody - ChIP-seq Grade, H3K4me3 Antibody - ChIP-seq Grade (sample size).
I used ChIP-qPCR with the Diagenode CRISPR/Cas9 polyclonal antibody to successfully show that Cas9 binds to the target region of my sgRNA, validating my CRISPR experiment. The antibody produced minimal background signal at non-specific genomic regions. I am now using the antibody to validate further sgRNA in different CRISPR cell lines.
ChIP was performed on Jurkat cells expressing dCas9-VP64-mCherry and a sgRNA targeting the IL1RN promoter. Each IP was performed using 4 million cells and 2 µL CRISPR/Cas9 polyclonal antibody (Diagenode C15310258) or 1 µg rabbit IgG control antibody (Diagenode C15410206). qPCR was carried out on undiluted ChIP DNA using SYBR green and PCR primers directed against the sgRNA binding site at IL1RN, as well as two non-target regions at the SLC4A1 and TP53 promoters. ChIP enrichment was measured using the percent input method.Researcher from the University of Manchester
about CRISPR/Cas9 Antibody - ChIP-seq Grade, CRISPR/Cas9 Antibody - ChIP-seq Grade (sample size).
Diagenode’s CRISPR/Cas9 polyclonal antibody shows superior signal than the original clone 7A9: a researcher from EPFL in Lausanne, Switzerland has compared these two antibodies in Western blot.
Western blot was performed using HCT116 DKO cells transduced with Krab-dCas9 (2) or non-transduced (1) cells. Then, 100,000 cells were lysed in sample buffer 2x and boiled 5 min at 95°C before loading in a 15% acrylamide gel. The same sample was loaded 3x in the same gel. The membrane was cut in 3 parts for each antibody. Membrane was blocked 1h with 3% BSA at RT. Antibodies were diluted 1:1,000 in 3% BSA and incubated overnight at 4°C. Secondary incubation was done for 1h at RT (1:10,000 dilution). Anti-hnRNPA1 was used as a loading control.EPFL in Lausanne
about CRISPR/Cas9 Antibody - ChIP-seq Grade, CRISPR/Cas9 Antibody - ChIP-seq Grade (sample size).
As Genomic services facility, Genoma Lab team, have a large experience, processing different samples for NGS library prep and usually those protocols start with a fragmentation step and frequently the enzimatic option works fine at the first attemp, for the majority kind of samples, Some complex samples (environmental and clinical samples) are more resistant and require more treatments with different setups, which is a problem in low amount DNA and unique samples, causing at the same time a negative impact in experimental planning, times of processing samples and cost. Because of this, the introduction of mechanical fragmentation strategy, using Diagenoder One, became more relevant in the Lab routine. Bioruptor One improved our NGS workflows, providing better quality, cost and time of experiments, with a quick, easy, versatile and clean method!Ph.D. Carolina Sánchez & Genoma Lab team. Genomic services facility Universidad Mayor. Santiago. Chile.
about Diagenode One sonication device, 50 ?l Microfluidic Chips for the One.
We have had an excellent experience with the RRBS service provided by Diagenode. Our project was based on DNA extracted from human fresh and paraffin-embedded skeletal muscle, and the RRBS and Bioinformatic results were reliable and consistent with what we expécted. We highly recommend their serviceMario Roque - Instituto de Histología y Embriología de Mendoza (IHEM), Mendoza, Argentina
about RRBS Service (Reduced Representation Bisulfite Sequencing).
We are very happy with the services provided by Diagenode. In our project, Reduced Representation Bisulfite Sequencing (RRBS) was used and it worked well for both high-quality DNA and DNA extracted from formalin-fixed, paraffin-embedded (FFPE) material. Bioinformatic analyses were comprehensive and high-quality results were obtained. Diagenode’s services can be recommended.Satu Mäki-Nevala, PhD, University of Helsinki
about RRBS Service (Reduced Representation Bisulfite Sequencing).
We sheared the DNA on the Diagenode One and used the MicroPlex Library Preparation v2 Kit to create DNA libraries for whole genome sequencing of four plant species for which there is no reference genome available. Previous attempts with a commercial Tn5-transposase based method gave unsatisfactory results. However, the Diagenode MicroPlex kit was quicker, easier, and gave the expected profile of fragment sizes. In just 30 seconds of sonication, we obtained a fragment distribution centered at 270 bp. The library construction took only 2 hours with this kit. The library was sequenced in a NexSeq 550 in High-Output mode, giving 85% based with>Q30.PhD. Ricardo Verdugo, Assistant Professor, University of Chile
about Diagenode One sonication device, MicroPlex Library Preparation Kit v2 (12 indexes), MicroPlex Library Preparation Kit v2 (12 indexes).
I like the Diagenode CATS RNA-seq kit for a number of reasons.Jordan Lewandowski, PhD; HSCRB // Harvard
The kit is approachable for any research user. The things I care about are time, reproducibility, and ease. In my hands, the CATS mRNA-seq kit has generated highly reproducible libraries from very low input RNA that goes into polyA selection. This has significantly decreased the amount of time I spend doing library preps, enabling me to further engage with my research.
about CATS mRNA-seq Kit (with polyA selection) v2 x24, CATS mRNA-seq Kit (with polyA selection) v2 x12, CATS mRNA-seq Kit (with polyA selection) v2 x96.
Given the low input range of up to 1 ng of our cell free plasma derived small RNA samples, commercially available kits and various published protocols failed in generating a library suitable for NGS with attempts resulting in high concentrations of adaptor dimer or failed library amplifications.
In light of this, we are more than happy to have tested the CATS small RNA-seq kit by Diagenode. Even with limited input material we got a rich library with absolutely no detectable adaptor dimer, as expected given it is a ligation free method. The kit performed superbly, in contrast to other commercially available kits or published protocols, with less hassle and with a greater ease of use.
Highly recommended for anyone who is struggling with generating a library from low input and challenging samples.
Jonatan Darr, PhD, Environmental Epigenetics Group, Institute of Experimental Genetics, Helmholtz Zentrum München, Germany
about CATS Small RNA-seq Kit x24, CATS Small RNA-seq Kit x12, CATS Small RNA-seq Kit x96.
I have used Diagenode's antibodies for my ChIP Seq experiments. The antibodies performed very well in our experiments with specific signal, and good signal to noise ratio.
Antibodies used in our lab: Pol II Monoclonal Classic (C15200004).Junaid Akhtar, Institute of Development Biology and Neurobiology, Mainz, Germany
about Pol II Antibody - ChIP-seq Grade, Pol II Antibody - ChIP-seq Grade (sample size).
Established in March 15th 2000, DNA Link, Inc. has developed as one of the leading full genomic service provider with its extensive 17 years of experience. DNA Link has been involved in numerous genomic researches including many national projects, and contributed to the expansion of the genomic industry.
On one hand, DNA Link was the very first company to introduce the RS II system from Pacific Bioscience in Asia, and has been approved as one of the eight global certified service providers for PacBio. Since 2012, DNA Link has been providing quality results that are controlled under strict criteria and successfully assisted numerous research projects.
PacBio platforms’ strongest upside is that it generates the longest reads among all sequencing platforms, and constructing long-read libraries using high molecular weight DNA is the most crucial part of utilizing the platforms. Within the PacBio library preparation process, shearing the DNA molecules accordingly to the library size is the initial step for a successful library construction. Sufficient library yield can be obtained only if the DNA molecules are sheared within the targeted size.
DNA Link has been using the Megaruptor instrument from Diagenode for this shearing process, as it is easy to use and has less contamination risk for using disposable hydropores, which contributes to the overall efficiency of the laboratory logistics.
about Megaruptor® 2.
I have been using Diagenode products to perform ChIP-seq during the last three years and I am very satisfied, with the Bioruptor, the kits and the antibodies. I have used the iDeal ChIP-seq kit for Histones and the iDeal ChIP-seq kit for Transcription Factors with very successful and reproducible results. Once I tried to ChIP histones with a home-made protocol and it worked much worse in comparison with Diagenode kits. In other occasion, I tried a non-Diagenode antibody for a transcription factor and I also got much poor results, however with the Diagenode antibody I always got very nice results. I strongly recommend the use of Diagenode products.Dr. Francisca Martinez Real - Development and Disease Research Group - Max Planck Institute for Molecular Genetics, Berlin, Germany
about iDeal ChIP-seq kit for Histones, iDeal ChIP-seq kit for Histones, iDeal ChIP-seq kit for Histones, iDeal ChIP-seq kit for Transcription Factors, iDeal ChIP-seq kit for Transcription Factors, iDeal ChIP-seq Kit for Transcription Factors.
Neonatal screening cards provide a valuable bio-bank for retrospective studies. We have used the Bioruptor Pico and IP-Star Compact instrument along with the Auto MeDIP and IPure v2 kit to generate genome-wide DNA methylation profiles using only minute DNA inputs. Using the Diagenode instruments and products has generated reproducible results and saved us hands-on time. We are satisfied with the level of service and technical support and would recommend Diagenode to all others working in the field of epigenetics.
about Bioruptor® Pico sonication device, IP-Star® Compact Automated System, IPure kit v2.
The new Diagenode Premium RRBS Kit makes it easy to use RRBS cost-effectively and with high throughput, using early sample pooling and multiplex sequencing. Most importantly, the method provides an improved coverage of up to 4 million CpGs for the human genome. We successfully used this protocol on more than 1,000 samples comprising of six different species, various cancers, FFPE and lowinput samples.Paul Datlinger and Christoph Bock, CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
about Premium Reduced Representation Bisulfite Sequencing (RRBS) Kit, Premium Reduced Representation Bisulfite Sequencing (RRBS) Kit, RRBS Service (Reduced Representation Bisulfite Sequencing).
I have been doing ChIPs for a very long time and have tried many kits from different sources like Active Motif, Millipore/Upstate, and homemade reagents. The reproducibility and binding efficacy were never optimal for these until a colleague recommended the iDeal ChIP-seq Kit for Transcription Factors from Diagenode. I have done more than one hundred samples of ChIPs and ChIP-seq using this kit. The results are very consistent and the binding efficacy is higher than with all the other methods. I would definitely recommend this ChIP kit from Diagenode to anyone who is trying to do ChIP or ChIP-seq.Researcher at Johns Hopkins University, School of Medicine
about iDeal ChIP-seq kit for Transcription Factors.