Reduced Representation Bisulfite Sequencing (RRBS) Services

Species	No. of reads aligned (alignment rate)	No. of unique CpGs covered (alignment rate)	Bisulfite conversion rate (%)	Mean sequencing depth per covered CpG (×)
Human	21,787,346 (74%)	3,913,287	99.4	14
Human FFPE	27,290,117 (79%)	2,525,053	99.7	28
Rat	27,499,313 (86%)	1,663,104	99.0	38
Dog	4,053,009 (84%)	3,572,384	99.4	9
Zebrafish	29,035,877 (83%)	1,685,466	99.3	48

QC of the genomic DNA	• Measurement of DNA concentration using the Qubit (Thermo Fisher) • Assessment of the DNA quality using the Fragment Analyzer (Advanced Analytical)
Preparation of RRBS libraries	• MspI digestion • Library preparation (ends preparation, adaptor ligation) • Size selection • Sample pooling • Bisulfite conversion • Library amplification and clean-up • QC of the RRBS library pool (DNA concentration, analysis of the pool profile)
Deep sequencing	• Samples are sent for sequenced on an Illumina platform, Single-Read, read length 50 bp (SR50) • 40 million pre-filtered reads (average) are obtained per sample when pooling 8 samples/lane • Detection of methylation status of 3-4 million CpGs on average (human) • Mean CpG coverage is 10-15x for human samples when pooling 8 samples/lane

Analysis	Features
Standard	Methylation calling: alignment of bisulfite sequencing data to reference genome and determination of methylation status of CpG nucleotides.
Annotation in genomic regions	Annotation of differentially methylated CpGs or of DMRs with genomic regions such as introns, exons, enhancers (when available), promoters, intergenic regions.
Differential methylated regions analysis (DMR)	Comparison of methylation status of CpG nucleotides between sample groups.
Gene ontology terms analysis	Enrichment analysis on gene sets. Gene Ontology terms that are overrepresented in differentially bound regions may indicate the underlying biological processes involved.
Pathway analysis	Identify biochemical pathways in which genes associated with differentially methylated regions (or individual differentially methylated CpGs) may be overrepresented

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 service
Mario Roque - Instituto de Histología y Embriología de Mendoza (IHEM), Mendoza, Argentina

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

Our laboratory has used RRBS sevice of Diagenode on murine and human samples. The service was impeccable in each phase, from the sample preparation to bionformatic analysis because it was always customer-oriented. I highly recommend my colleagues to use the RRBS service from Diagenode.
Prof. Lucia Altucci, MD, PhD, Seconda Università degli Studi di Napoli, Dipartimento di Biochimica, Biofisica e Patologia generale .

Diagenode Premium RRBS technology: Cost-effective DNA methylation mapping with superior CpG resolution and coverage APPLICATION NOTE DNA methylation is an important epigenetic mark with broad relevance in development and disease. ...	Download
The Diagenode Epigenetics custom service POSTER Complete workflows for genome-scale DNA methylation and histone marks analysis Epigenetics is cr...	Download
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How to properly cite this product in your work

Diagenode strongly recommends using this: DNA Methylation Profiling (RRBS Service) (Diagenode Cat# G02020000). Click here to copy to clipboard.

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Demethylation of ITGAV accelerates osteogenic differentiation in a blast-induced heterotopic ossification in vitro cell culture model.
Logan NJ, Camman M, Williams G, Higgins CA
Trauma-induced heterotopic ossification is an intriguing phenomenon involving the inappropriate ossification of soft tissues within the body such as the muscle and ligaments. This inappropriate formation of bone is highly prevalent in those affected by blast injuries. Here, we developed a simplified cell culture mod...

Paternal sepsis induces alterations of the sperm methylome and dampens offspring immune responses—an animal study
Katharina Bomans, Judith Schenz, Sandra Tamulyte, Dominik Schaack, Markus Alexander Weigand and Florian Uhle
Abstract Background Sepsis represents the utmost severe consequence of infection, involving a dysregulated and self-damaging immune response of the host. While different environmental exposures like chronic stress or malnutrition have been well described to reprogram the germline and subsequently offspring a...

DNMT3B overexpression contributes to aberrant DNA methylation and MYC-driven tumor maintenance in T-ALL and Burkitt’s lymphoma
Poole et al.
Aberrant DNA methylation is a hallmark of cancer. However, our understanding of how tumor cell-specific DNA methylation patterns are established and maintained is limited. Here, we report that in T-cell acute lymphoblastic leukemia (T-ALL) and Burkitt’s lymphoma the MYC oncogene causes overexpression of DNA me...

Diagenode® Premium RRBS technology: cost-effective DNA methylation mapping with superior coverage
Anne-Clémence Veillard, Paul Datlinger, Miklos Laczik, Sharon Squazzo & Christoph Bock
Reduced representation bisulfite sequencing (RRBS) enables genome-scale DNA methylation analysis in any vertebrate species. The assay benefits from the practical advantages of bisulfite sequencing while avoiding the cost of whole-genome sequencing. The Diagenode Premium RRBS kit makes this technology widely availabl...

DNA Methylation Profiling (RRBS Service)

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