Liang et al. BMC Genomics 2011, 12:231 http://www.biomedcentral.com/1471-2164/12/231 stages of development was particularly striking. Even though proliferative changes in cell populations may underlie many apparent methylation differences during development, the results still indicate that extensive dif- ferences in methylation status are associated with devel- opment. Although the majority of the methylation differences were associated with non-CpGi promoter regions, there were also many methylation differences associated with non-promoter regions that may have novel roles in regulating development. Methods Growth of ES cells and collection of tissues, DNA and RNA preparations ES cells were grown as previously described [32,53]. C57 BL/6J mice were purchased from Jackson Laboratory and maintained less than three generations in the department of Laboratory Animal Research facility at Roswell Park Cancer Institute under an approved IACUC protocol. Conventional pair breeding was performed, the plug was confirmed and then the male mouse was removed from the breeding cage after one day. Pregnant mice were sacri- ficed for embryo collection 15 days later (E15). Embryonic sacs were kept in PBS solution on ice. After the quick con- firmation of development stage, embryos were subjected to dissection under the microscope (Leica MZ-125). Brain, heart, liver and testis tissues were carefully obtained from male embryos (verified later, see following). Male new born (NB) mice were collected within 24 hours after deliv- ery and used for collection of the same tissues. 12 weeks old adult male mice (AD) were also used for adult tissue collection as described before [32]. Tissues and cells were immediately snap-frozen in liquid nitrogen and stored at - 80 °C until use. The sex of embryonic mice was determined by using Y chromosome marker and the Zfxy genotyping. PCR was performed to confirm sex by using primers (Zfxya-’: AGCTGTTTCATAGTCACAGAACTTAC, Zfy-’-11: CGAATGTGATGACTGTAGGAAGAATC, and Zfx-5’-2: AGAAAGCCATAGAATGCGATGAGTGC). PCR results showing multiple bands indicated male mice, which were then used in this study. Mixtures of 6 male mouse tissues were used for DNA isolation for E15 and NB analysis since it was not feasible to obtain enough DNA from a sin- gle E15 or NB animal. Adult tissues were collected from two separate animals. DNA was then prepared from 2 separate mixtures (E15 and NB) and 2 separate adults. Genomic DNA from all tissue samples was extracted using the DNeasy Kit (QIAGEN) according to Qiagen protocol. Sequenom MassARRAY quantitative methylation analysis Sequenom MassARRAY quantitative methylation analysis was performed as previously described [32] using Page 12 of 16 protocols provided by Sequenom (see Additional file 13 for a list of primers used). All oligonucleotides were pur- chased from Integrated DNA Technologies (Coralville, IA). Amplification of 1 μl bisulfite-treated DNA (~20 ng/ ml) was performed using HotStar Taq Polymerase (Qia- gen) in a 5 μl reaction volume using PCR primers at a 200 nM final concentration. PCR amplification was per- formed with the following parameters: 94 °C for 15 min hot start, followed by denaturing at 94 °C for 20 s, annealing at 56 °C for 30 s, extension at 72 °C for 1 min for 45 cycles, and final incubation at 72 °C for 3 min. PCR products were processed for MassARRAY analysis according to the manufacturer’s instructions (Sequenom hMC) by the Microarray and Genomics Core Facility at Roswell Park Cancer Institute. In the mass spectrum, the relative amount of methylation can be calculated by com- paring the signal intensity between mass signals of methylated and non-methylated template DNA. Each spot is ionized 50× per second at five different rasters with all resultant methylation calls performed by the Epi- Typer software v1.0 (Sequenom). All data is transferred and stored in an Oracle database for tabulation. A mini- mum of two independently derived tissue DNAs were analyzed. Methylated DNA Immunoprecipitation analysis: MeDIP [33] High-quality genomic DNA isolated from E15, NB and AD mouse tissues, was digested with MseI (TTAA) (New England Biolabs) to produce small fragments (200 - 1,000 bp) while keeping CpGi regions intact. Fragmen- ted DNA was heat denatured to produce single-stranded DNA, and a portion of the denatured DNA stored as control (input) DNA. Monoclonal mouse anti 5-methyl cytidine ( Eurogentec) was used to immunoprecipitate methylated DNA fragments. The immune complexes were captured with Protein A Agarose beads (Invitro- gen). Complexes were washed to remove nonspecifically bound material. Following elution of bound complexes, ethanol precipitation, and resuspension of MeDIP DNA, a small aliquot of DNA and control input DNA were used to amplify a known methylated DNA region and a known unmethylated DNA region by real-time PCR. The primers of Pst4, Pst6 and Pst21 that were identified by RLGS previously [32] were used in enrichment mea- surements according to the manufacturer’s instructions (NimbelGen). Ratios between the MeDIP and input values are defined and normalized against a known unmethylated control sequence. After validating the enrichment of MeDIP DNA, MeDIP DNA and control input DNA were amplified by whole-genome amplifica- tion kit (Sigma Aldrich), followed by purification (QIA- GEN Quick PCR Purification Kit) and then sent to NimbleGen for Microarray hybridization according to their standard protocol for the array.