Deoxyribonuclease digestion

Placental dimensions were obtained after delivery by alignment of the longest axis of the placenta (length) on a grid and recording the widest point perpendicular to the longest dimension axis (width). Placental tissue was collected from four representative locations (1 cm³ each), washed, and immersed into PBS with protease and phosphatase inhibitors for protein analysis or RNAlater for gene expression analysis (Thermo Fisher Scientific, Waltham, MA, USA). Samples were processed according to manufacturer’s instructions and stored at −80°C until further analysis. Collection time and storage temperature were recorded until samples were shipped to the University of Colorado, Denver, USA. Total RNA was isolated from villous placenta stored in RNAlater using a combination of TRI reagent (Molecular Research Center, Cincinnati, OH, USA) and RNeasy-mini columns, including on-column deoxyribonuclease digestion (Qiagen, Germantown, MD, USA) (64 (link), 65 (link)). Directional cDNA libraries for mRNA-sequencing were prepared using polyA-mRNA from individual RNA samples using Illumina TruSeq reagents (64 (link), 65 (link)). Detailed methods are described in Supplementary Materials. Pooled cDNA libraries were sequenced (150 bp paired reads) using a NovaSeq 6000 instrument (Illumina, San Diego, CA, USA). Data analysis is described in the statistical analyses section.