The Epigenomics Profiling Core is pleased to announce that it now offers cfMeDIP (circulating cell-free methylated DNA immunoprecipitation) as part of its DNA methylation analysis repertoire.
Contact Dr. Marcos Estecio to learn how this service can help advance your DNA methylation-related research projects. (email@example.com)
ATAC-seq and CUT&RUN
We offer ATAC-seq (Assay for Transposase Accessible Chromatin with high-throughput sequencing) to assess genome-wide chromatin accessibility, transcription factor binding, and nucleosome positioning and CUT&RUN (Cleavage Under Targets and Release Using Nuclease) for genome-wide profiling of DNA-bound proteins.
Contact Dr. Abhinav Jain for details. (firstname.lastname@example.org)
DNA Methylation Analysis Services
The Epigenomics Profiling Core is funded, in part, through the Department of Epigenetics and Molecular Carcinogenesis and is open to researchers across MD Anderson and in the Texas Medical Center. The core is located at the MD Anderson Cancer Center Basic Sciences Research Building. The DNA Methylation Analysis arm of the core is a fully equipped molecular biology laboratory with dedicated instrumentation including a Qiagen Pyrosequencing machine, an Illlumina MiSeq instrument, and an Oxford Nanopore Technologies GridION sequencer.
The main obstacle to DNA methylation analysis is that methylated cytosines cannot be detected simply by sequencing. During PCR amplification, methylated cytosines are not differentiated by the DNA polymerase and like unmethylated cytosines, they are paired with guanosine dinucleotides. Thus, capturing methylated cytosines depends on indirect methods. The most commonly used methods are (1) restriction enzyme-based approaches, which take advantage of methylation-sensitive enzymes; (2) affinity-based approaches, in which either antibodies against 5-methylcytosine or against methyl-binding domain proteins are used to collect the methylated fraction of the genome, and (3) bisulfite conversion of non-methylated cytosines to thymidine through a hydrolytic deamination reaction, which takes advantage of the non-reactivity of methylated cytosines to free hydroxyl groups. Each one of these methods has an important application in studying the epigenome. Among these methods, bisulfite conversion is the gold-standard due to its high resolution when combined with sequencing methods. In this way, every single cytosine can be identified as methylated or unmethylated.
DNA Methylation Analysis Workflow
Our workflow begins with a researcher-initiated request for consultation and service. Depending on whether a project will investigate the methylation state of an individual gene or the genome one of two workflows is applied:
1) Candidate gene assays are designed and optimized as needed. DNA sample integrity and quality are assessed before bisulfite treatment. Treatment is followed by amplification of the specific region(s) of interest and pyrosequencing.
2) For genome-wide studies, reduced representation bisulfite sequencing (RRBS) is applied to questions regarding promoters and CpG islands, whereas whole genome bisulfite sequencing (WGBS) is used to answer more global questions. For samples with limited material (under 10 ng of genomic DNA), we use cfMeDIP-seq to investigate genome-wide 5-methylcytosine or 5-hydroxymethylcytosine semi-quantitatively. Once sample quality has been assured, it is either enzymatically digested or sonically sheared into appropriately sized pieces. Adaptors are then ligated onto the DNA ends before treatment with bisulfite. Following treatment, sequencing libraries are prepared and checked for quality before sending them to a next-generation sequencing facility.
Consultation and Bioinformatics
The choice of an appropriate DNA methylation analysis method depends on whether the investigation is still in the discovery stage or whether a set of targets has already been decided. Further, special considerations regarding assay design and detection methods must be addressed when dealing with minimal amounts of DNA or when applying DNA methylation as a molecular marker. The DNA Methylation Analysis Facility will work with investigators to design assays and conduct experiments. To arrange a consultation, please contact Marcos Estécio, Ph.D., at email@example.com.
Researchers with projects that involve next-generation sequencing services should always consult a bioinformatician before planning the detailed project. For assistance with bioinformatics analyses, MD Anderson users may consult with their institutionally assigned, departmental bioinformatician, seek advice on potential bioinformatics collaborators from the Epicore directors.
Pyrosequencing DNA Methylation Analysis (PMA)
After bisulfite-conversion, gene-specific targets are amplified in a PCR reaction with primers that produce amplicons from both the methylated and unmethylated alleles. Selected CpG sites are quantified in a pyrosequencing reaction. The advantages of this method are sensitive and quantitative reading of methylation with medium to high throughput.
In the image to the right, a region near the transcription start
site of the gene HAND1 was selected for assay design based on prior
microarray experiments. The amplified region covered four CpG sites,
which are highlighted in yellow. In the example, the colon cancer cell
line RKO showed high levels of DNA methylation at all examined CpG
sites (average = 97%), but the same gene is unmethylated in normal
peripheral blood lymphocytes (6.3%).
In bisulfite sequencing, as in pyrosequencing, gene-specific targets are amplified by PCR using primers that will produce amplicons from both methylated and unmethylated alleles. Sequencing of individually cloned products by Sanger sequencing can reveal the methylation status of dozens of sites and identify allelic-specific methylation.
Reduced Representation Bisulfite Sequencing (RRBS)
A combination of enzyme-based and bisulfite-based methods, RRBS is one the most popular genome-wide methods used today due to its ability to accommodate various starting amounts of DNA. DNA is digested with a restriction enzyme and selected for size. Post-adapter ligation ensures enrichment for CpG islands. The DNA is then bisulfite-treated and amplified with universal primers. The resulting RRBS libraries are checked for quality and then transferred to a next-generation sequencing facility for final processing. In general, between 400,000 and 1 million CpG sites are evaluated in a single experiment.
The image shows a genome browser view of RRBS data for the prostate cell lines RWPE-1 and Du145. Each vertical bar shows the methylation density of one CpG site, ranging from 0% to 100%. A differentially methylated region (DMR) is shown overlapping the CpG island present at the alternative transcriptional start site of the TP73 gene.
Circulating Cell-Free Methylated DNA Immunoprecipitation (cfMeDIP)
In this method, antibodies that specifically recognize 5-methylcytosine (5mC)- or 5-hydroxymethylcytosine (5hmC)-modified bases in DNA are used to selectively capture genomic fragments carrying these modifications and deplete non-modified fragments. The use of barcoding in library preparations allows for multiplexed sequencing of samples for next-generation sequencing. The methods are compatible with DNA amounts ranging from 5 ng to 100 ng, and potentially even lower amounts, depending on DNA quality. This method is suitable for evaluating circulating-cell free DNA collected from plasma or serum, as well as genomic DNA obtained from formalin-fixed, paraffin-embedded (FFPE) samples.
The images on the left illustrate the specificity of anti-5hmC antibodies. When applied to sequences with unmodified cytosines and modified cytosines, only 5hmC-marked DNA is capture with over a thousand-fold enrichment. The Genome Browser view and correlation graphic highlight the reproducibility of cfMeDIP-seq to detected 5hmC when using different starting amounts of DNA (5ng and 100ng) and compared to publicly available data for the same cell line.
Whole Genome Bisulfite Sequencing (WGBS)
WGBS is currently the only truly whole-genome approach for DNA methylation analysis of the entire human genome and covers approximately 90% of all CpG sites. In this method, the entire genome is fragmented by sonication, and modified adaptors are ligated to the DNA fragments prior to bisulfite-conversion. Then, as in RRBS, the amplified DNA is subjected to high-throughput sequencing.
The genome browser view on the right shows WGBS data compared to RRBS data for TP73 in the prostate cell lines RWEP-1 and Du145. Multiple differentially methylated regions are visible across the gene body.
Chromatin Analysis Services
The Epigenomics Profiling Core is funded, in part, through the Department of Epigenetics and Molecular Carcinogenesis and is open to researchers across MD Anderson and in the Texas Medical Center. The core is located at the MD Anderson Cancer Center Basic Sciences Research Building. The Chromatin Analysis arm of the core is ready to serve your Chromatin Immunoprecipitation (ChiP), ChIP-seq, CUT&RUN (Cleavage Under Targets and Release Using Nuclease), and ATAC-seq (Assay for Transposase Accessible Chromatin with high-throughput sequencing) needs.
Each new project starts with a personal consultation between the researcher and Dr. Abhinav Jain (firstname.lastname@example.org) to determine the most suitable assay for the project, all appropriate controls, and the choice of antibodies and oligos for quality control (QC) assessments of experimental samples. Dr. Jain will provide project-specific guidelines for sample preparation, sample requirements and submitting samples to the EpiCore. He will stay in communication with investigators to provide project updates and experiment results. Dr. Jain is also available to assist with data interpretation upon request. Researchers with projects that involve next-generation sequencing (NGS) should always consult a bioinformatician before planning the detailed project.
Chromatin Immunoprecipitation (ChIP) Services
ChIP followed by Next Generation Sequencing (NGS) is a major tool used to profile both epigenetic changes and the enrichment of chromatin and DNA binding factors across the genome. In addition to traditional ChIP-seq, the EpiCore also offers histone ChIP-seq assays in a high throughput format (HT-ChIP), which aids simultaneous screening of samples with multiple antibodies targeting individual transcription factors and/or specific histone modifications with minimal sample-to-sample variation.
EpiCore ChIP services include all steps outlined below, starting from cells or tissues.
- Prepare high-quality chromatin: successful ChIP experiments require high-quality chromatin that we will prepare from frozen pellets of fixed cells or from flash-frozen tissues (please contact us for sample preparation guidelines). Chromatin integrity is assured through quality control (QC) measures taken prior to proceeding to ChIP
- Pull-down of protein-bound DNA using either antibodies recognizing specific histone post-translational modifications (PTMs), available at EpiCore (appropriate negative and positive control antibodies are included to determine the quality of the ChIP assay) , or user preferred custom antibodies*
- Detect enrichment of the protein of interest at a specific genomic locus (ChIP-qPCR)
- Prepare libraries for next-generation sequencing to detect enrichment of the protein of interest genome-wide (ChIP-Seq). After performing QC measures, the EpiCore will submit the prepared libraries to the user preferred NGS resource for sequencing
- Perform assessments throughout the process for QC
The following histone PTM antibodies, commonly used to identify specific genomic features (e.g. promoters, enhancers, heterochromatin and gene bodies), are available through the EpiCore:
- H3K4me3 and H3K27me3 (promoters)
- H3K4me1 and H3K27ac (enhancers)
- H3K9me3 (heterochromatin)
- H3K36me3 and H3K79me2 (gene bodies/active transcription)
*Investigator-preferred antibodies can also be used. These must be provided by the customer and should have been previously validated for ChIP. The EpiCore has successfully performed ChIP-Seq with more than 50 investigator-supplied, non-histone antibodies.
CUT&RUN (Cleavage Under Targets and Release Using Nuclease) is a revolutionary, new, cost- and labor-effective, technique to determine the chromatin occupancy of a specific protein with fewer cells (~500,000 in most cases) and fewer sequencing reads than traditional ChIP-seq methods. In addition, CUT&RUN does not require a cross-linking step, and overcomes the problem of fragmentation bias.
In CUT&RUN, the protein of interest is targeted by an antibody specific for that protein. The antibody is then targeted by a recombinant protein A-micrococcal nuclease fusion that cuts and releases DNA fragments from bulk chromatin at the sites of antibody binding. CUT&RUN services at the EpiCore include the steps listed below, starting from a sample of cells.
- Harvest the required number of cells and isolating nuclei, as required
- Pull-down protein-bound DNA using either antibodies recognizing specific histone post-translational modifications (PTMs) available at the EpiCore (appropriate negative and positive control antibodies are included to determine the quality of the CUT&RUN assay) or user preferred custom antibodies*
- Prepare libraries for next-generation sequencing to detect enrichment of protein of interest genome-wide. After QC the EpiCore will submit the libraries to the user-preferred NGS core for sequencing
- Perform assessments at discrete points throughout the process for QC
*Investigator-preferred antibodies can be used. These must be provided by the customer and should have been previously validated. Please discuss antibody and control requirements with Dr. Jain.
The EpiCore offers full service ATAC-seq (Assay for Transposase Accessible Chromatin with high-throughput sequencing) services. ATAC-seq helps customers address scientific questions regarding chromatin accessibility, transcription factor binding, and nucleosome positioning across the genome with reduced mitochondrial DNA contamination compared to more traditional methods. The ATAC-seq services offered at the EpiCore can be performed with fewer cells than MNase-seq, DNase-seq and FAIRE-seq typically require. Our services include all of the processing and quality control (QC) steps listed below, starting from cells/tissues.
- Harvest the required number of cells and isolate nuclei
- Perform the transposition reaction
- Prepare DNA library from open chromatin to profile chromatin accessibility
- Assess enrichment of open chromatin by qPCR at validated genomic locations using primers pre-designed by EpiCore
- Perform assessments at discrete points throughout the process for QC
After consultation with Dr. Jain (email@example.com), please request Chromatin Analysis services via iLab