Time-course small RNA sequencing across photoperiods in Arabidopsis thaliana

Small RNAs play important roles in photoperiodism, but a comprehensive characterization of miRNAs and the more recently recognized tRFs within this context is lacking. Herein, we characterize the patterns of miRNAs and tRFs in Arabidopsis by performing time-course sRNA-sequencing across three photoperiods. By comparing with our previous mRNA-sequencing time-courses (GSE221846), we identified 14 negatively-correlated miRNA-mRNA pairs between the two co-sequenced datasets that suggest photoperiodic sRNA regulations. Furthermore, we identified 20 patterns of photoperiodic tRFs. These patterns are linked to the tRNA isotypes and positions they derive from, suggesting that tRFs are subjected to photoperiodic regulations. Overall design: Seeds were sterilized before being sown onto 1/2 Murashige and Skoog medium plates lined with autoclaved filter papers. For germination, seeds were stratified in the dark for 48 hours before being transferred to a growth chamber under a 12L:12D photoperiod . After germination, seedlings were kept in the same condition for 10 days. On day 11, the seedlings were transferred to 16L:8D, 12L:12D, or 8L:16D photoperiod. On day 13, whole seedlings including shoots and roots were harvested and snap-frozen in liquid nitrogen.

小RNA（small RNAs）在光周期调控中发挥关键作用，但目前仍缺乏针对该调控背景下微小RNA（miRNAs）以及近年被发现的tRNA衍生片段（tRFs）的系统性表征研究。本研究以拟南芥为材料，通过对三种光周期条件下的样本进行时序小RNA测序（sRNA-sequencing），系统性解析了其体内miRNAs与tRFs的表达模式。通过与本团队前期发表的时序mRNA测序（mRNA-sequencing）数据集（GSE221846）进行比对，我们在这两个时序测序数据中共鉴定出14对呈负相关的miRNA-mRNA调控对，提示存在光周期依赖的小RNA调控机制。此外，我们还鉴定出20种光周期响应型tRFs表达模式，这些模式与其来源的tRNA亚型及衍生位点密切相关，表明tRFs同样受到光周期调控。整体实验设计：将种子灭菌后，点播于铺有高压灭菌滤纸的1/2 Murashige和Skoog（MS）培养基平板上。播种后，种子先于黑暗条件下层积处理48小时，随后转移至光照周期为12小时光照/12小时黑暗的生长箱中萌发。萌发后，幼苗在该条件下继续培养10天。至第11天时，将幼苗分别转移至16小时光照/8小时黑暗、12小时光照/12小时黑暗以及8小时光照/16小时黑暗三种光照周期条件下。至第13天时，收取包含地上部分与根系的整株幼苗，立即置于液氮中速冻保存。