Placenta sample collection and DNA methylation quantification
STERILE, PAT, CLOTS
Placentas obtained at delivery were rinsed with sterile saline, pat dried with paper towel, and had nonadherent clots removed. The placental membrane and umbilical cord were trimmed before biopsies were taken. Four biopsies measuring 0.5 cm × 0.5 cm × 0.5 cm were collected directly below the fetal surface of each placenta within 1 h of delivery. The samples were placed in RNALater and frozen at – 80 °C for molecular analysis. The placental biopsy samples were processed at the Columbia University Irving Medical Center as described previously [Standard Illumina protocols were followed for background correction, normalization to internal control probes, and quantile normalization. The Illumina 450k array’s plating scheme was adjusted according to the assay’s internal QC design. The GenomeStudio QC standard was implemented during data preprocessing, and the internal probes have been used for background correction, dye bias correction, normalization, probe-design bias correction, and an offset for Infinium I and II probe intensity. The assay quality controls comprised of controls for measuring staining sensitivity and controls for testing efficiency of bisulfite conversion. Bisulfite modification was performed using the EZ Methylation kit (Zymo Research, CA). Bisulfite-converted sequences without CpGs served as negative controls; the mean of the negative control probes was used as the system background. The resulting intensity files were processed with Illumina’s Genome Studio which generated average beta values for each CpG site (i.e., the fraction of methylated sites per sample by calculating the ratio of methylated and unmethylated fluorescent signals) and detection Missing CpGs were imputed by the k-nearest neighbors method, setting 