Research Institute for Food and Agriculture, Ryukoku University, Yokotani, Seta Oe-cho, Otsu, Shiga, Japan.
Center for Ecological Research, Kyoto University, Hirano, Otsu, Shiga, Japan.
Sci Rep. 2019 May 8;9(1):7091. doi: 10.1038/s41598-019-43600-0.
RNA-Seq is a whole-transcriptome analysis method used to research biological mechanisms and functions but its use in large-scale experiments is limited by its high cost and labour requirements. In this study, we have established a high-throughput and cost-effective RNA-Seq library preparation method that does not require mRNA enrichment. The method adds unique index sequences to samples during reverse transcription (RT) that is conducted at a higher temperature (≥62 °C) to suppress RT of A-rich sequences in rRNA, and then pools all samples into a single tube. Both single-read and paired-end sequencing of libraries is enabled. We found that the pooled RT products contained large amounts of RNA, mainly rRNA, causing over-estimations of the quantity of DNA and unstable tagmentation results. Degradation of RNA before tagmentation was found to be necessary for the stable preparation of libraries. We named this protocol low-cost and easy RNA-Seq (Lasy-Seq) and used it to investigate temperature responses in Arabidopsis thaliana. We analysed how sub-ambient temperatures (10-30 °C) affected the plant transcriptomes using time-courses of RNA-Seq from plants grown in randomly fluctuating temperature conditions. Our results suggest that there are diverse mechanisms behind plant temperature responses at different time scales.
RNA-Seq 是一种全转录组分析方法，用于研究生物机制和功能，但由于其成本高和劳动力需求大，在大规模实验中的应用受到限制。在本研究中，我们建立了一种高通量且具有成本效益的 RNA-Seq 文库制备方法，不需要进行 mRNA 富集。该方法在更高的温度（≥62°C）下进行逆转录（RT）时，在样本中添加独特的索引序列，以抑制 rRNA 中富含 A 的序列的 RT，然后将所有样本汇集到一个管中。该方法可以进行单读和双端测序文库。我们发现，汇集的 RT 产物中含有大量的 RNA，主要是 rRNA，导致 DNA 数量的高估和不稳定的标签化结果。我们发现，在标签化之前，RNA 的降解对于文库的稳定制备是必要的。我们将这个方案命名为低成本且简便的 RNA-Seq（Lasy-Seq），并使用它来研究拟南芥的温度响应。我们通过对在随机波动温度条件下生长的植物进行时间序列的 RNA-Seq 分析，研究了亚环境温度（10-30°C）如何影响植物的转录组。我们的结果表明，在不同的时间尺度上，植物对温度的响应有多种机制。
