RNA sequencing of the Murine Oviduct Infundibulum. 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.

针对小鼠皮下植入Alzet渗透式微型泵（Alzet osmotic minipump）进行长期催乳素（prolactin）处理（3 μg/小时/只，持续7天）后，小鼠漏斗部（infundibulum）的基因表达变化显示，催乳素可下调纤毛（cilium）发育与功能所需的基因。综合上述结果可知，催乳素可调控漏斗部纤毛细胞功能的周期性变化；同时该结果还揭示了催乳素长期升高可通过额外机制对生育能力产生负面影响。

整体实验设计：实验动物分为两组，分别给予重组小鼠催乳素（prolactin，以下简称PRL）或生理盐水对照处理，持续7天。处死动物后解剖取出漏斗部，立即置于液氮中速冻以用于RNA提取。每只小鼠的两侧漏斗部组织合并为一份样品，再将两只小鼠的样品进一步合并（即每个RNA测序样本包含4个漏斗部组织，每组处理设置5个样本）。采用Illumina NovaSeq 6000测序平台开展全转录组测序：对样本中的信使RNA（mRNA）进行链特异性富集，随后采用100碱基对（base pair, bp）双端（paired end, PE）测序读长（PE100）进行测序，每个样本产出约8000万条读段。使用FastQC质量评估软件对测序读段进行质量质控，随后通过Trimmomatic工具以质量阈值15及Truseq接头序列进行读段剪切，丢弃长度短于20bp的读段。采用剪接感知型短读段比对工具（Hisat2、Samtools）将过滤后的读段比对至小鼠mm10参考基因组。使用FeatureCounts工具完成原始读段计数定量，再通过DESeq2工具开展两组间差异比较，以筛选对照组与催乳素处理组间的差异表达基因。