RNA sequencing of the Murine Oviduct Infundibulum

Changes in gene expression in the infundibulum after a prolonged prolactin treatment (3 µg/h/mouse, 7 days, subcutaneous Alzet osmotic minipump) showed prolactin down regulation of genes necessary for cilium development and function. Collectively, these results suggest that prolactin regulates cyclic changes in ciliated cell function in the infundibulum. They also suggest an additional mechanism whereby prolonged prolactin elevation would negatively impact fertility. Overall design: Animals were treated with recombinant murine PRL or saline as a control for 7 days. Infundibula were dissected and immediately snap frozen for RNA extraction. Both infundibular regions were pooled per mouse and two animals were pooled for each RNAseq sample (4 infundibula per sample, 5 samples per treatment). Whole transcriptome sequencing was performed using the Illumina NovaSeq 6000 system. Samples were enriched for mRNA (stranded) and sequenced by 100 base pair (bp) paired end (PE) reads (PE100, 80 million reads per sample). Reads were quality assessed using FastQC quality assessment software and trimmed using a quality threshold of 15 and Truseq adapter sequences (reads shorter than 20bp were discarded, Trimmomatic). Reads were aligned to the mouse mm10 reference genome with a splice aware short read aligner (Hisat2, Samtools). FeatureCounts was used to quantify raw counts and two group comparison, using DESeq2 to identify differentially expressed genes between control and PRL-treated animals.