DNA methylation analysis

DNA was isolated from decidua-free placentas at E12.5 and submitted to EpigenDx for site-specific methylation analysis. DNA methylation analysis of the Igf2/H19 imprint control center was performed by EpigenDx (http://www.epigendx.com/).

High quality genomic DNA was isolated from E12.5 decidua-free placentas (n=3) using standard phenol chloroform extraction and ethanol precipitation. RRBS was performed by the Epigenomics core at WCMC as follows: i) 5, 50 or 1000 ng of high quality genomic DNA were digested with 200 U of MspI (NEB R0106S) in a 100 ml reaction for up to 16 hours at 37°C. DNA was isolated using standard phenol chloroform extraction and ethanol precipitation and resuspended into 10 mM Tris pH 8.0.ii) End repair of digested DNA was performed using 15 U of T4 DNA polymerase (NEB M0203L), 5 U of Klenow DNA polymerase (NEB M0210L), 50 U of T4 Polynucleotide Kinase (NEB M0201L), 4 ml of premixed nucleotide triphosphates each at 10 mM (NEB N0447L) using T4 DNA ligase buffer with 10 mM dATP (NEB B0202S). The reaction was incubated at 20°C for 30 minutes and products were isolated using QIAquick PCR purification columns (Qiagen). iii) Adenylation was performed using 15 U Klenow fragments (NEB M0212L) and 10 ml of dATP at 1 mM concentration. The reaction was incubated at 37°C for 30 minutes and products were isolated using MinElute PCR purification columns (Qiagen). iv) Adenylated DNA fragments were ligated with pre-annealed 5-methylcytosine-containing Illumina adapters using 2000 U T4 DNA ligase (NEB M0202T) and 1.2 mM final concentration of methylated adapters at 16°C for 16 hours. Products were isolated using MinElute columns (Qiagen). v) Library fragments of 150–175 and 175–225 bp run on 1.5% low range ultra agarose gel were isolated using the Qiaquick Gel Extraction kit (Qiagen). vi) Bisulfite treatment was performed using the EZ DNA Methylation Kit (Zymo Research). vii) PCR amplification for each library was performed using FastStart Hifidelity DNA Polymerase (Roche) and PCR products were isolated using AMPure XP beads (Agencort). viii) All amplified libraries underwent quality control steps including using Qubit 1.0 fluorometer and a Quant-iT dsDNA HS Assay Kit for quantitation (Invitrogen) and bioanalyzer visualization (Agilent 2100 Bioanalyzer). Bioinformatics analysis was performed by the Epigenomic core at WCMC.