CELLULAR REPROGRAMMING Volume 16, Number 3, 2014 Original Article ª Mary Ann Liebert, Inc. DOI: 10.1089/cell.2014.0002 Methylated DNA Immunoprecipitation and High-Throughput Sequencing (MeDIP-seq) Using Low Amounts of Genomic DNA Ming-Tao Zhao,1 Jeffrey J. Whyte,1 Garrett M. Hopkins,2 Mark D. Kirk,2 and Randall S. Prather1 Abstract DNA modifications, such as methylation and hydroxymethylation, are pivotal players in modulating gene ex- pression, genomic imprinting, X-chromosome inactivation, and silencing repetitive sequences during embryonic development. Aberrant DNA modifications lead to embryonic and postnatal abnormalities and serious human diseases, such as cancer. Comprehensive genome-wide DNA methylation and hydroxymethylation studies provide a way to thoroughly understand normal development and to identify potential epigenetic mutations in human diseases. Here we established a working protocol for methylated DNA immunoprecipitation combined with next-generation sequencing [methylated DNA immunoprecipitation (MeDIP)-seq] for low starting amounts of genomic DNA. By using spike-in control DNA sets with standard cytosine, 5-methylcytosine (5mC), and 5- hydroxymethylcytosine (5hmC), we demonstrate the preferential binding of antibodies to 5mC and 5hmC, respectively. MeDIP-PCRs successfully targeted highly methylated genomic loci with starting genomic DNA as low as 1 ng. The enrichment efficiency declined for constant spiked-in controls but increased for endogenous methylated regions. A MeDIP-seq library was constructed starting with 1 ng of DNA, with the majority of fragments between 250 bp and 600 bp. The MeDIP-seq reads showed higher quality than the Input control. However, after being preprocessed by Cutadapt, MeDIP (97.53%) and Input (94.98%) reads showed comparable alignment rates. SeqMonk visualization tools indicated MeDIP-seq reads were less uniformly distributed across the genome than Input reads. Several commonly known unmethylated and methylated genomic loci showed consistent methylation patterns in the MeDIP-seq data. Thus, we provide proof-of-principle that MeDIP-seq technology is feasible to profile genome-wide DNA methylation in minute DNA samples, such as oocytes, early embryos, and human biopsies. Introduction CpG islands or non-CpG islands may cause embryonic re- tardation and serious diseases such as cancer. For example, NA methylation is one of the well-studied epigenetic aberrant DNA methylation of promoter CpG islands in tumor Dmarks that can be inherited during cell division. DNA suppressor genes is associated with silencing of their func- methylation is dynamically regulated during embryonic de- tions, which is considered as a ‘‘CpG island methylator velopment, and aberrant methylation changes are correlated phenotype’’ in many types of cancer (Baylin and Jones, with tumor development and other human diseases (Baylin 2011). Therefore, genome-wide DNA methylation analysis and Jones, 2011; Smith and Meissner, 2013). In mammals, opens a new avenue for thoroughly understanding normal DNA methylation is frequently present in the context of CpG, development and epigenetic origin of human diseases. albeit less frequent found in CpHpG (H = A, T, C). The ma- DNA methylation is established and maintained by DNA jority of the mammalian genome is composed of vast oceans methyltransferases (DNMT1, DNMT3a, and DNMT3b) that where sparse but highly methylated CpG dinucleotides (non- are essential for embryonic and postnatal development in CpG islands) are distributed and isolated CpG islands with a mammals(Lietal.,1992;Okanoetal.,1999).However, high density of CG content that are resistant to DNA DNA methylation information is lost during standard mo- methylation (Deaton and Bird, 2011). Epimutations in either lecular biology manipulations, such as molecular cloning 1Division of Animal Sciences and 2Division of Biological Sciences, University of Missouri, Columbia, MO, 65211. 1 2 ZHAO ET AL. in bacteria and PCRs, due to lack of maintenance of DNA Materials and Methods methyltransferases. Therefore, several approaches have mESC culture, embryoid body production, been proposed to preserve DNA methylation information and neural differentiation and simultaneously transform it into quantitative and mea- surable signals (Laird, 2010). Of them, the most prevailing mESCs with a transgene for enhanced green fluorescent techniques include methylated DNA immunoprecipitation protein (eGFP) (B5 cell line) were cultured as adherent (MeDIP), bisulfite sequencing (BS-seq), and reduced rep- colonies in the presence of leukemia inhibitory factor (LIF; resentation bisulfite sequencing (RRBS). Combined with Chemicon), as previously described (Meyer et al., 2004). high-throughput, next-generation sequencing, these tech- All chemicals are from Sigma (St. Louis, MO, USA) unless niques have provided comprehensive genome-wide infor- indicated otherwise. Briefly, undifferentiated ESCs were mation regarding DNA methylation. In terms of the merit thawed and grown in gelatin-treated T25 tissue culture and bias of these methods, BS-seq and RRBS can generate a flasks in ESC growth medium plus LIF (ESGM; LIF at base-resolution DNA methylome, whereas MeDIP-seq can 1000 U/mL), including Dulbecco’s modified Eagle medium only generate relative enrichment of specific regions across (DMEM; Invitrogen), 10% newborn calf serum, 10% fetal the genome. Nevertheless, bisulfite treatment inevitably bovine serum (FBS), nucleosides, and b-mercaptoethanol causes substantial DNA degradation and thus requires mi- (1 mM). Typically, to maintain the undifferentiated state crogram levels of DNA Input. In addition, the bisulfite during proliferation, ESC cultures were refreshed every conversion efficiency is also a concern, even though many other day with ESGM plus fresh LIF and passaged from one commercial kits claim more than 99% conversion. Special to two T25 flasks. For future use, adherent ESC cultures enzymes, such as uracil-tolerant DNA polymerase, are were dissociated with 0.25% trypsin; the trypsin was in- usually employed for high-throughput library preparation in activated with ESGM followed by a slow freeze to -80°C BS-seq. and transferred to liquid nitrogen for long-term storage. Affinity-enrichment methods such as MeDIP were ini- Otherwise, neural differentiation was achieved by using the tially combined with microarray technology to compare the 4- /4+ protocol that includes exposure to retinoic acid for genome-wide DNA methylation among samples (Weber neural induction (Bain et al., 1995). Briefly, the ESCs were et al., 2005). Subsequently, MeDIP was combined with a cultured in ESC induction medium (ESIM; ESGM minus next-generation sequencing platform to investigate the LIF and b-mercaptoethanol) in uncoated Petri dishes for 4 DNA methylome in precious samples, such as bone marrow days and grown as suspended embryoid bodies (EBs). After cells, mammalian oocytes, primordial germ cells, and 4 days in ESIM, the majority of ESCs formed floating preimplantation embryos. The MeDIP-seq protocol has spheres called EBs that had started neural differentiation. been optimized to reach as low as 50 ng of DNA for starting On day 4 of neural induction, the EBs were transferred into materials (Taiwo et al., 2012). Borgel et al. performed ESIM plus 500 nM all-trans retinoic acid (Sigma) and cul- MeDIP followed by whole-genome amplification and mi- tured for an additional 4 days to complete the neural in- croarray hybridization on early-stage mouse embryos duction phase. Finally, the EBs were collected, snap frozen starting with 150 ng of DNA (Borgel et al., 2010). The in liquid nitrogen, and subsequently stored at -80°C until sixth DNA modification 5-hydroxymethylcytosine (5hmC) genomic DNA extraction. can also be detected genome-wide by using a hydro- xymethylated DNA immunoprecipitation (hMeDIP) pro- Methylated DNA immunoprecipitation cedure with a 5hmC-specific antibody (Ficz et al., 2011). Comparison between MeDIP-seq and hMeDIP-seq data Genomic DNA was extracted by using an AllPrep DNA/ helps better understand the distribution of 5-methylcytosine RNA Mini Kit (Qiagen) following the manufacturer’s (5mC) and 5hmC signals on the same genomic loci across instructions. The DNA concentration was measured with the whole genome. Recently, the MeDIP-seq method a NanoDrop spectrophotometer (Thermo Scientific). The has been applied to detect other DNA modifications, such as DNA was then randomly sheared into fragments with an 5-formylcytosine and 5-carboxylcytosine (Shen et al., average size of 400 bp by an M220 focused-ultrasonicator 2013), thus illustrating the powerful advantages of MeDIP- (Covaris). The MeDIP procedure was modified according to Seq technology on genome-wide DNA modification a standard protocol with low starting amounts of DNA studies. (Borgel et al., 2012). To prevent DNA loss during the The traditional MeDIP-seq requires microgram levels of MeDIP procedure, special low DNA LoBind tubes (Ep- starting DNA that may not be applied to minute DNA pendorf ) were used. Initially sonicated DNA (1 lg, 100 ng, samples, such as mammalian oocytes and preimplantation 10 ng, and 1 ng) was diluted in 450 lL of TE buffer (10 mM embryos. Here we took advantage of next-generation se- Tris-HCl, 1 mM EDTA, pH 8.0) and then denatured at 95°C quencing technology to profile genome-wide DNA methyl- for 10 min. A portion of sonicated DNA was reserved as ation by using 1 ng of genomic DNA from mouse embryonic Input that did not undergo immunoprecipitation. Denatured stem cells (mESCs). After index