GSM1646425: sample_G; Homo sapiens; Bisulfite-Seq
In the Danish Mole Project, samples from conceptuses presenting with vesicular villi on ultrasound or by visual inspection of the aborted tissue, are collected consecutively. The histopathology is revised blindly with respect to the results of the genetic analyses. The Danish Mole Biobank contains 309 samples collected in the period April 1986-June 2003. At the histopathological revision 270 were classified as originating from hydatidiform moles. The genetic constitution was analyzed by karyotyping, flow cytometric assessment of the nuclear DNA contents, and analysis of polymorphic DNA markers. 162 HMs were diploid, of which 151 were diploid androgenetic (AnHM) whereas 11 showed biparental DNA markers. Eight of the diploid moles with biparental markers were mosaics, in that two different cell lines were identified, a biparental cell line and an androgenetic cell line (AnHM/BiHM), whereas three of the 11 moles contained cells with biparental genomes, only (BiHM) (20,21). RRBS libraries based on single MspI digestion were constructed for the normal placentas and the HMs/HM-like placentas described above. Briefly, 100 ng of genomic DNA was digested with 300U of MspI enzyme (NEB) in 100 μl reactions at 37°C for 16–19 h. The digested DNA was purified by MiniElute PCR Purification Kit (Qiagen). To prepare the samples for the ligation of 5-methylcytosine modified adapters, the overhangs generated by digestion were repaired in 100 μl reactions containing 15 units of T4 DNA polymerase, 2 units of Klenow fragment, 60 units of T4 polynucleotide Kinase, 0.6 mM of dNTP and 1× polynucleotide kinase buffer. Reactions were incubated at 20°C for 30 min, followed by purification. The blunt ended DNA fragments were 3’ adenylated using 15 units of Klenow (3’→5’ exo-) (Enzymatics) and 0.2 mM of dATP in 50 μl reactions with 1× blue buffer (Enzymatics) at 37°C for 30 min.   Purification using a MiniElute PCR Purification Kit (Qiagen) was performed and the products were resolved in 50 μl reaction mixture containing 360 units of T4 DNA ligase, 0.12 μM of 5-methylcytosine modified adapter oligo mix (5’GATCGGAAGAGCACACGTCTGAACTCCAGTCAC and 5’ TACACTCTTTCCCTACACGACGCTCTTCCGATCT) in 1× Rapid ligation buffer, and incubated at 20°C for 15 min, then 65°C for 15 min. A 2% of agarose gel was used to separate the fractions of adapter-ligated fragments. To obtain DNA fragments of 40-120 bp and 120-220 bp genomic DNA, 160-240 bp and 240-340 bp adapter ligated fragments were excised from the gel and purified using a QIAquick gel extraction kit (Qiagen). Bisulfite conversion was conducted using a ZYMO EZ DNA Methylation-Gold Kit™ (ZYMO), following the manufacturer’s instructions. Finally, the libraries were generated by PCR amplification in a reaction volume of 50 μl consisting of 10 μl of bisulfite converted fragments, 4 μl of 2.5 mM dNTP, 5 μl of 10× PCR buffer, 0.5 μl of JumpStart™ Taq DNA Polymerase (Sigma), 2 μl of PCR primers and 28.5 μl of water. The following thermal cycling program was used: 94°C/1 min; 11 to 15 cycles of 94°C/10 s, 58°C/30 s and 72°C/30 s; extention for 5 min at 72°C and the products were kept at 12°C. The PCR products were size-selected and purified using a QIAquick gel extraction kit (Qiagen). To assess the C-T conversion rate of the bisulfite treatment, 50 pg of ummethylated lambda DNA was added to the genomic DNA samples. The libraries were analyzed by an Agilent 2100 Bioanalyzer (Agilent Technologies).