RNA sequencing and Ingenuity® Pathway Analysis

Total RNA was extracted from E19 male placentae (Control n = 2 and Obese n = 3), as previously outlined. One microgram of total RNA was depleted of ribosomal RNA and PolyA tails of coding RNAs were captured by treatment with Oligo-dT beads. Complementary DNA (cDNA) libraries were generated after an amplification step, according to the TruSeq Stranded Total RNA Sample Preparation Guide (Illumina, San Diego, CA, USA), and quantified using KAPA Library Quantification Kit. Multiplex single-read sequencing was performed using Illumina HiSeq 2500 (Illumina). Sequence reads were demultiplexed using the CASAVA pipeline (Illumina) and then aligned to the Mus musculus genome (GRCm38) using TopHat version 2.0.11. Raw read counts and fragments per kilobase of transcript per million mapped reads (FPKM) were generated using Cufflinks version 2.2.1. A quality check of mapped reads was executed using the R package CummeRbund. Databases were trimmed for exclusion of very low detection or undetectable genes. The resulting data were analyzed using edgeR by calculating the likelihood ratio, and by adjusting P values via Benjamini and Hochberg’s method to control the false discovery rate (FDR). Ingenuity Pathway Analysis (IPA) was applied to identify biological pathways related to the genes that were differentially expressed between Control and Obese E19 male placentae. The placenta RNA sequencing (RNA-seq) data have been deposited in GEO database under the accession number {"type":"entrez-geo","attrs":{"text":"GSE140013","term_id":"140013"}}GSE140013.