Preparation of Reduced Representation Bisulfite Sequencing (RRBS) libraries (Prot 70)
DNA methylation is a well-known epigenetic mark consisting in the addition of a methyl group to the cytosine producing the 5-methylcytosine (5mC). 5mC is abundant in mammalian genomes and occurs almost exclusively at CpG dinucleotides. Globally, mammalian genomes are CpG-poor but some regions, termed CpG islands, have a high CpG frequency and are found frequently in promoters. Only 2 to 4% of CpG islands are methylated in somatic cells. This process is essential to maintain stable gene repression of developmental, imprinted and X-linked genes. Several methods are available for mapping DNA methylation. Compared to the Whole Genome Bisulfite Sequencing, the Reduced Representation Bisulfite Sequencing (RRBS) allows a good coverage of gene promoters and CpG islands for a lower sequencing cost (Meissner et al. 2008). Here we describe an optimized RRBS protocol, derived from the original protocol described by (Gu et al. 2011), which allows the use of small amount of starting DNA (2ng) with good efficiency.
1- CNRS, University of Strasbourg, UMR7242 Biotechnology and Cell, signalling 300, Bd Sébastien Brant - 67412 Illkirch, France
Corresponding author: Michael Weber
Nano-MeDIP-seq: Methylome analysis using low DNA concentrations (Prot 58)
To understand the functional consequences of DNA methylation on phenotypic plasticity, a genome-wide analysis should be embraced. This in turn requires a technique that balances accuracy, genome coverage, resolution and cost, yet is low in DNA input to minimise the drain on precious samples. MeDIP-seq fulfils these criteria, combining methylated DNA immunoprecipitation (MeDIP) with massively-parallel DNA sequencing. Methylated DNA Immunoprecipitation (MeDIP) is a technology capable of targeting the vast majority of the methylome. It involves antibodies directed against mC/mCG to precipitate methylated DNA fragments. MeDIP is able to detect methylated cytosines in both mC and mCG contexts. Because antibodies used for MeDIP were raised in a way to yield equal specificity against mC and mCG, MeDIP offers a near-unbiased and hypothesis-free approach without a priori assumptions about which regions of the methylome might be targeted (see below). Combining MeDIP with next generation sequencing (MeDIP-seq; Down et al., 2008), provides high-quality methylomes at typically 100-300bp resolution (depending on chosen insert size) at costs comparable to other capture-based techniques (Beck, 2010). In this EpiGeneSys Protocol Collection, which is based in an original publication in Nature Protocols (Taiwo et al., 2012), we detail Nano-MeDIP-seq – a protocol that uses 100-fold less genomic DNA than that which is commonly used for immunoprecipitation-based applications. Applications of this method will result in specific and sensitive enrichment of methylated DNA fragments over a wide range of DNA concentrations (5,000-50 ng), making Nano-MeDIP-seq suitable for studies involving minute clinical samples, micro-dissected tissues and rare cell types.
1 UCL Cancer Institute, University College London, London WC1E 6BT, UK.
2 UCL Institute of Healthy Ageing, University College London, London WC1E 6BT, UK
3 Laboratory of Developmental Genetics and Imprinting, The Babraham Institute, Cambridge CB22 3AT, UK
4 Centre for Trophoblast Research, University of Cambridge, Cambridge CB2 3EG, UK
Corresponding author: Oluwatosin Taiwo, Stephan Beck
Methyltransferase-based single-promoter analysis assay (Prot 38)
Nucleosomal positioning [reviewed in (1-3)] plays a pivotal role in the regulation of transcriptional initiation. Transcriptional co-activator complexes interact with nucleosomes (4) to induce nucleosomal rearrangements. Nucleosomes often have to unfold completely (5) or be disassembled (6) at the transcription start site, to allow for transcriptional initiation (7, 8).
Most of the studies done on the nucleosomal rearrangements of histones use conventional footprinting techniques, which rely on nuclease digestion and primer extension. However, promoters are molecular ‘modules’, which are controlled as individual entities. When analyzed by conventional methodologies this modularity is destroyed. Our lab has modified a previously described footprinting strategy (9,10), which now allows us to study the chromatin structure of individual molecules. MSPA (methylation-sensitive promoter analysis) allows for the study of unmethylated CpG islands by treatment of nuclei with the CpG-specific DNA methyltransferase SssI (M.Sssi), followed by genomic bisulfite sequencing of individual progeny DNA molecules (11-13). This gives single molecule resolution over the promoter and allows for the physical linkage between binding sites on individual promoter molecules to be maintained.
Our lab has successfully used this method to study the difference in nucleosomal positioning in the p16 promoters in two human cell lines (11), to identify transcription factor binding sites and their combinatorial organization during endoplasmic reticulum stress (12), and to study the changes in nucleosome occupancy silencing of the three transcription start sites in the bidirectional MLH1 promoter CpG island in cancer cells (13).
University of Southern California - Norris Comprehensive Cancer Center - 1441 Eastlake Ave
Los Angeles, CA 90033, USA
Bisulfite sequencing of very small samples (Prot 37)
(based on Olek et al., 1996, Schoenherr et al., 2003)
DNA methylation is a stable epigenetic mark, which can mediate gene silencing. Bisulfite sequencing allows for precise identification of methylated cytosines within DNA (Frommer et al. 1992). This method is based on different rate of chemical conversion of methylated and non-methylated cytosines to uracil where non-methylated cytosines are converted efficiently while methylated cytosines remain non-reactive. This method was further developed by embedding analyzed DNA into an agarose bead (Olek et al. 1996). The protocol presented here was further optimized for bisulfite sequencing of small samples where the starting material was a small number of cells (Fedoriw et al. 2004; Svoboda et al. 2004). The smallest amount of material from which several unique clones were recovered was 25 oocytes, which corresponds to 100 DNA molecules in the initial material (Svoboda et al. 2004). This protocol can be also used for analyzing up to 200ng purified genomic DNA in one sample.
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic
Videnska 1083 - 142 20 Prague 4, Czech Rebulic
Bisulfite sequencing of small DNA/cell samples (Prot 35)
Sequencing of sodium-bisulfite modified genomic DNA originally introduced by M. Frommer (Frommer et al., 1992) is a widely used “gold standard” method for DNA-methylation analysis. Since this method relies on a harsh chemical treatment of DNA it causes a lot of DNA damage and hence a dramatic loss of high quality DNA for PCR amplification and further analysis. In the meantime several commercial kits are available for this procedure which work reasonably well when starting with large amounts of DNA.
Here we describe a protocol for small numbers of cells and little DNA which requires some specific handling. The protocol is based on a strategy originally introduced by our lab (Hajkova et al., 2002) using agarose embedded DNA. This physical trapping helps to avoid DNA loss during the various incubation steps while maintaining a good bisulphite conversion rate. We will introduce two alternative procedures to perform bisulphite treatment of agarose embedded small DNA aliquots or cells and guide through some generally critical points in the bisulphite reaction and primer design. We also include tips for the process of data processing after sequencing which is facilitated by a new and very useful software tool (BiQ Analyzer). This tool allows rapid and reproducible processing and evaluation of bisulphite sequencing data. It generates standardized table output formats allowing direct database integration.
Saarland University, FR 8.3 Biosciences
Dept. of Genetics/Epigenetics
Campus Saarbrücken - 66123 Saarbrücken, Germany