RRBS Service (Reduced Representation Bisulfite Sequencing)

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RRBS Service

Reduced representation bisulfite sequencing (RRBS) enables genome-scale DNA methylation analysis at the single nucleotide level in any vertebrate species. The assay benefits from the practical advantages of bisulfite sequencing while avoiding the cost of whole genome sequencing. By cutting the genome using the restriction MspI enzyme (CCGG target sites) followed by size selection, DNA is enriched to represent biologically relevant target CpG-rich regions including promoters and CpG islands. Our RRBS service makes this technology widely available and provides high coverage (up to 7 million CpGs detected in human samples).

Genome-scale DNA methylation analysis

  • Robust and cost-effective solution with reliable results
  • Capability to use gDNA inputs down to 25 ng
  • Use of UDIs enhance sequencing results
  • UMI technology eliminates PCR duplicates
  • High coverage - up to 7 million CpGs detected in human samples
  • Single nucleotide resolution
  • Detection of methylation patterns in CpG rich regions including promoters and CpG islands
  • Suitable for any vertebrate species

Complete end-to-end service

  • From DNA QC to sequencing raw data
  • Leveraging our Premium RRBS kit V2 widely adopted
  • As little as 25 ng for any vertebrate species
  • Differentially methylated site analysis using bioinformatics tools

See our other DNA Methylation Profiling Services


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
  • Services Workflow

    RRBS Service includes:

    QC of the genomic DNA
    • Measurement of DNA concentration 
    • Assessment of DNA quality
    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 sequenced on an Illumina platform, paired-end reads, read length 50 bp (PE50)
    • 40 million raw reads (on average) per sample when pooling 10 samples/lane
    • 7 million CpGs (on average) for human samples
    • 7-11x CpG coverage (on average) for human samples
  • Bioinformatics Analysis



    • FASTQ raw data
    • FASTQC quality control insights
    • Alignment of bisulfite sequencing data against reference genome
    • Methylation calling and extraction
    • Summary statistics
    Differential methylation analysis
    • Methylation level analysis
    • Differentially Methylated CpGs (DMCs) analysis
    • Differentially Methylated Regions (DMRs) analysis
    • Annotation of DMCs and DMRs for genomic regions (exons, introns, …)
    • Clustering analysis

    Gene ontology terms analysis

    • Enrichment analysis on gene associated with DMCs and DMRs
    • Get functional insights

    Pathway analysis

    • Identification of biological pathways in which genes associated with DMCs and DMRs may be over-represented (or under-represented)
    • Get mechanistic insights
  •  Testimonials

    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 .
  •  Documents
    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. ...
    The Diagenode Epigenetics custom service POSTER
    Complete workflows for genome-scale DNA methylation and histone marks analysis Epigenetics is cr...
    Epigenomics Profiling Services FLYER
    Chromatin analysis DNA methylation services RNA-seq analysis
  •  Publications

    How to properly cite this product in your work

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    Using our products in your publication? Let us know!

    Long-term effects of myo-inositol on traumatic brain injury: Epigenomic and transcriptomic studies
    Oganezovi N. et al.
    Background and purpose Traumatic brain injury (TBI) and its consequences remain great challenges for neurology. Consequences of TBI are associated with various alterations in the brain but little is known about long-term changes of epigenetic DNA methylation patterns. Moreover, nothing is known about potential trea...

    Gestational Caloric Restriction Alters Adipose Tissue Methylome and Offspring’s Metabolic Profile in a Swine Model
    Mas-Pares B. et al.
    Limited nutrient supply to the fetus results in physiologic and metabolic adaptations that have unfavorable consequences in the offspring. In a swine animal model, we aimed to study the effects of gestational caloric restriction and early postnatal metformin administration on offspring’s adipose tissue epigene...

    Diagnostic Algorithm to Subclassify Atypical Spitzoid Tumors in Low and High Risk According to Their Methylation Status
    Gonzales-Munoz J.F. et al.
    Current diagnostic algorithms are insufficient for the optimal clinical and therapeutic management of cutaneous spitzoid tumors, particularly atypical spitzoid tumors (AST). Therefore, it is crucial to identify new markers that allow for reliable and reproducible diagnostic assessment and can also be used as a predi...

    Sperm DNA methylation is predominantly stable in mice offspring bornafter transplantation of long-term cultured spermatogonial stem cells.
    Serrano J. B.et al.
    BACKGROUND: Spermatogonial stem cell transplantation (SSCT) is proposed as a fertility therapy for childhood cancer survivors. SSCT starts with cryopreserving a testicular biopsy prior to gonadotoxic treatments such as cancer treatments. When the childhood cancer survivor reaches adulthood and desires biological chi...

    Myelodysplastic Syndrome associated TET2 mutations affect NK cellfunction and genome methylation.
    Boy M. et al.
    Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders, representing high risk of progression to acute myeloid leukaemia, and frequently associated to somatic mutations, notably in the epigenetic regulator TET2. Natural Killer (NK) cells play a role in the anti-leukemic immune response via their cytolyti...

    Epigenetics and stroke: role of DNA methylation and effect of aging onblood-brain barrier recovery.
    Phillips C. et al.
    Incomplete recovery of blood-brain barrier (BBB) function contributes to stroke outcomes. How the BBB recovers after stroke remains largely unknown. Emerging evidence suggests that epigenetic factors play a significant role in regulating post-stroke BBB recovery. This study aimed to evaluate the epigenetic and trans...

    Altered DNA methylation in estrogen-responsive repetitive sequences ofspermatozoa of infertile men with shortened anogenital distance.
    Stenz L. et al.
    BACKGROUND: It has been suggested that antenatal exposure to environmental endocrine disruptors is responsible for adverse trends in male reproductive health, including male infertility, impaired semen quality, cryptorchidism and testicular cancer, a condition known as testicular dysgenesis syndrome. Anogenital dist...

    Epigenetic Alterations of Repeated Relapses in Patient-matchedChildhood Ependymomas.
    Zhao Sibo et al.
    Recurrence is frequent in pediatric ependymoma (EPN). Our longitudinal integrated analysis of 30 patient-matched repeated relapses (3.67 ± 1.76 times) over 13 years (5.8 ± 3.8) reveals stable molecular subtypes (RELA and PFA) and convergent DNA methylation reprogramming duri...

    DNA methylation may affect beef tenderness through signal transduction inBos indicus.
    de Souza M. M. et al.
    BACKGROUND: Beef tenderness is a complex trait of economic importance for the beef industry. Understanding the epigenetic mechanisms underlying this trait may help improve the accuracy of breeding programs. However, little is known about epigenetic effects on Bos taurus muscle and their implications in tenderness, a...

    Genome-Wide Epigenomic Analyses in Patients With Nociceptive and Neuropathic Chronic Pain Subtypes Reveals Alterations in Methylation of Genes Involved in the Neuro-Musculoskeletal System
    Stenz et al
    Nociceptive pain involves the activation of nociceptors without damage to the nervous system, whereas neuropathic pain is related to an alteration in the central or peripheral nervous system. Chronic pain itself and the transition from acute to chronic pain may be epigenetically controlled. In this cross-sectional s...

    Genome-wide epigenomic analyses in patients with nociceptive andneuropathic chronic pain subtypes reveals alterations in methylation ofgenes involved in the neuro-musculoskeletal system.
    Stenz Ludwig et al.
    Nociceptive pain involves the activation of nociceptors without damage to the nervous system, whereas neuropathic pain is related to an alteration in the central or peripheral nervous system. Chronic pain itself and the transition from acute to chronic pain may be epigenetically controlled. In this cross-sectional s...

    Environmental enrichment preserves a young DNA methylation landscape in the aged mouse hippocampus
    Sara Zocher, Rupert W. Overall, Mathias Lesche, Andreas Dahl & Gerd Kempermann
    The decline of brain function during aging is associated with epigenetic changes, including DNA methylation. Lifestyle interventions can improve brain function during aging, but their influence on age-related epigenetic changes is unknown. Using genome-wide DNA methylation sequencing, we here show that experiencing ...

    Pathophysiological adaptations of resistance arteries in rat offspringexposed in utero to maternal obesity is associated with sex-specificepigenetic alterations.
    Payen Cyrielle et al.
    BACKGROUND/OBJECTIVES: Maternal obesity impacts vascular functions linked to metabolic disorders in offspring, leading to cardiovascular diseases during adulthood. Even if the relation between prenatal conditioning of cardiovascular diseases by maternal obesity and vascular function begins to be documented, little i...

    Muscle allele-specific expression QTLs may affect meat quality traitsin Bos indicus.
    Bruscadin J.J. et al.
    Single nucleotide polymorphisms (SNPs) located in transcript sequences showing allele-specific expression (ASE SNPs) were previously identified in the Longissimus thoracis muscle of a Nelore (Bos indicus) population consisting of 190 steers. Given that the allele-specific expression pattern may result from cis-regul...

    IGFBP2 protects against pulmonary fibrosis through inhibiting P21-mediated senescence
    Chiahsuan, C. et al.
    Accumulation of senescent cells contributes to age related diseases including idiopathic pulmonary fibrosis (IPF). Insulin-like growth factor binding proteins (IGFBPs) are evolutionarily conserved proteins that play a vital role in many biological processes. Overall, little is known about the functions of IGFBP2 in ...

    Perturbed DNA methylation by sustained overexpression of Gadd45b induces chromatin disorganization, DNA strand breaks and dopaminergic neurondeath in mice
    Ravel-Godreuil, C. et al.
    Heterochromatin disorganization is a key hallmark of aging and DNA methylation state is currently the main molecular predictor of chronological age. The most frequent neurodegenerative diseases like Parkinson disease and Alzheimer’s disease are age-related but how the aging process and chromatin alterations ar...

    Developmental cannabidiol exposure increases anxiety and modifiesgenome-wide brain DNA methylation in adult female mice.
    Wanner N. M. et al.
    BACKGROUND: Use of cannabidiol (CBD), the primary non-psychoactive compound found in cannabis, has recently risen dramatically, while relatively little is known about the underlying molecular mechanisms of its effects. Previous work indicates that direct CBD exposure strongly impacts the brain, with anxiolytic, anti...

    Hepatic transcriptome and DNA methylation patterns following perinataland chronic BPS exposure in male mice.
    Brulport A. et al.
    BACKGROUND: Bisphenol S (BPS) is a common bisphenol A (BPA) substitute, since BPA is virtually banned worldwide. However, BPS and BPA have both endocrine disrupting properties. Their effects appear mostly in adulthood following perinatal exposures. The objective of the present study was to investigate the impact of ...

    Integrative Analysis of Glucometabolic Traits, Adipose Tissue DNA Methylation and Gene Expression Identifies Epigenetic Regulatory Mechanisms of Insulin Resistance and Obesity in African Americans
    Neeraj K. Sharma, Mary E. Comeau, Dennis Montoya, Matteo Pellegrini, Timothy D. Howard, Carl D. Langefeld, Swapan K. Das
    Decline in insulin sensitivity due to dysfunction of adipose tissue (AT) is one of the earliest pathogenic events in Type 2 Diabetes. We hypothesize that differential DNA methylation (DNAm) controls insulin sensitivity and obesity by modulating transcript expression in AT. Integrating AT DNAm profiles with transcrip...

    Chronic cannabidiol alters genome-wide DNA methylation in adult mouse hippocampus: epigenetic implications for psychiatric disease.
    Wanner NM, Colwell M, Drown C, Faulk C
    Cannabidiol (CBD) is the primary non-psychoactive compound found in cannabis (Cannabis sativa) and an increasingly popular dietary supplement as a result of widespread availability of CBD-containing products. CBD is FDA-approved for the treatment of epilepsy and exhibits anxiolytic, antipsychotic, prosocial, and oth...

    LY75 Ablation Mediates Mesenchymal-Epithelial Transition (MET) in Epithelial Ovarian Cancer (EOC) Cells Associated with DNA Methylation Alterations and Suppression of the Wnt/β-Catenin Pathway.
    Mehdi S, Bachvarova M, Scott-Boyer MP, Droit A, Bachvarov D
    Growing evidence demonstrates that epithelial-mesenchymal transition (EMT) plays an important role in epithelial ovarian cancer (EOC) progression and spreading; however, its molecular mechanisms remain poorly defined. We have previously shown that the antigen receptor LY75 can modulate EOC cell phenotype and metasta...

    Obesogen effect of bisphenol S alters mRNA expression and DNA methylation profiling in male mouse liver
    Brulport Axelle, Vaiman Daniel, Chagnon Marie-Christine, Le Corre Ludovic
    Environmental pollution is increasingly considered an important factor involved in the obesity incidence. Endocrine disruptors (EDs) are important actors in the concept of DOHaD (Developmental Origins of Health and Disease), where epigenetic mechanisms play crucial roles. Bisphenol A (BPA), a monomer used in the man...

    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...

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