Time series RNA-seq of the Drosophila melanogaster female response to mating and Sex Peptide.. Time series RNA-seq of the Drosophila melanogaster female response to mating and Sex Peptide.

Sex Peptide, a seminal fluid protein of Drosophila melanogaster males, elicits an array of post-mating responses in females, including decreased receptivity to re-mating, and increased egg laying, activity and food intake, with a preference for protein-rich food. To determine how one protein can have such widespread effects, we set out to dissect the genetic architecture of the female’s response to Sex Peptide, to determine whether Sex Peptide alters the expression of several regulators targeted to specific post-mating responses or acts on a pleiotropic regulator that controls multiple responses. We performed bulk RNA-seq of female heads at 10 time points within the first 24 hours after mating, sampling virgin females, females mated to control males and females mated to Sex Peptide-null males. Using this high-resolution time series, we identified mating- and Sex Peptide-dependent differentially expressed genes and discovered the presence of differentially used exons. We constructed gene regulatory networks using clustering and motif enrichment analyses, and identified cell types in which these changes might take place using deconvolution of our bulk RNA-seq dataset. One key network included metabolic genes which might change in expression in the female’s fat body. A second network included genes with neuronal functions, whose changes might be located in neurons or sensory organs in the female’s head. Within these networks we identified known molecular regulators of the circadian clock. Further, we found that many differentially expressed genes, and some differentially used exons, followed a circadian rhythm in virgin females, and that this rhythm was altered after mating with a Sex Peptide- male. Overall design: Bulk RNA-seq was performed on the heads of D. melanogaster virign females, females mated to SP-null males and females mated to wildtype males. Samples were collected at 30 minutes and 1, 2, 3, 4, 5, 6, 8, 12 and 24 hours after mating. Virgin females were collected in parallel at the same time of day to avoid confounding of mating effects and circadian effects. The entire time series experiment was repeated 3 times, yielding 3 biological replicates.

性肽（Sex Peptide）是黑腹果蝇（Drosophila melanogaster）雄性精液中的一类分泌蛋白，可诱发雌性产生一系列交配后应答反应，包括降低再次交配的接受性、提升产卵量、活动水平与进食量，且表现出对高蛋白食物的偏好。为阐明单一蛋白何以能介导如此广泛的生理效应，本研究旨在解析雌性对性肽产生应答的遗传架构，以明确性肽是通过调控多种针对特定交配后反应的调节因子的表达，还是作用于控制多种反应的多效性调节因子。

我们在交配后24小时内的10个时间点对雌性头部开展批量RNA测序（bulk RNA-seq），采样对象涵盖未交配雌性、与对照雄性交配的雌性，以及与性肽缺失（Sex Peptide-null）雄性交配的雌性。依托这套高分辨率时间序列数据，我们鉴定出了依赖于交配与性肽的差异表达基因，并发现了差异使用外显子（differentially used exons）的存在。

我们通过聚类与基序富集分析构建了基因调控网络，并利用批量RNA测序数据集的反卷积分析，预测了这些表达变化可能发生的细胞类型。其中一个关键网络包含代谢基因，这类基因或在雌性脂肪体中发生表达改变；另一个网络则包含具有神经元功能的基因，其表达变化可能发生在雌性头部的神经元或感觉器官中。我们在这些网络中鉴定出了已知的生物钟（circadian clock）分子调节因子。此外，我们发现大量差异表达基因以及部分差异使用外显子在未交配雌性中呈现出昼夜节律，而在与性肽缺失雄性交配后，这种节律发生了显著改变。

整体实验设计：对黑腹果蝇的未交配雌性、与性肽缺失雄性交配的雌性，以及与野生型雄性交配的雌性的头部进行批量RNA测序。样本分别于交配后30分钟、1、2、3、4、5、6、8、12及24小时采集。同时在每日相同时间点采集未交配雌性样本，以消除交配效应与昼夜节律效应的混杂干扰。整套时间序列实验重复3次，共获得3个生物学重复样本。