Taxus cuspidata RNA-Seq analysis. Taxus cuspidata

This project is to analyze the over-wintering transcriptomic changes of Taxus cuspidata. Leaves (needles) of three north-facing branches and those of three south-facing branches of a male T. cuspidata tree (height: around 8 m) growing in front of the Institute of Low Temperature Science, Hokkaido University (43.1°N, 141.3°E) were harvested at around 10 am on September 24, 2019; October 24, 2019; November 5, 2019; December 3, 2019; December 25, 2019; February 25, 2020; March 25, 2020; April 23, 2020. RNA was extracted from these leaves and poly A+ RNA was further purified, and was analyzed with DNBSEQ. The fastP program (Chen et al. 2018) with default settings for paired-end reads was used to clean the RNA-seq reads. Subsequently, the Salmon program (Patro et al. 2017) was used to quantify the expression of the transcripts using the cleaned reads. The protein-coding region of the cDNAs obtained from the Iso-seq analysis were used as target sequences for the quantification. Transcripts per million (TPM) calculated by Salmon were used to normalize transcript read counts.

本项目旨在分析东北红豆杉（Taxus cuspidata）的越冬转录组变化。2019年9月24日、10月24日、11月5日、12月3日、12月25日，以及2020年2月25日、3月25日、4月23日每日上午10时左右，研究人员在北海道大学低温科学研究所（北纬43.1°，东经141.3°）前生长的一株株高约8米的雄性东北红豆杉上，分别采集3个北向枝条和3个南向枝条的叶片（针叶）。从采集的叶片中提取总RNA，进一步纯化得到poly A+ RNA，随后采用DNBSEQ平台进行测序分析。使用默认参数的fastP软件（fastP）对RNA-seq双端reads进行质控清洗，获得清洁测序读段。随后，使用Salmon软件（Salmon）基于清洁reads对转录本表达量进行定量，定量所用的靶序列为Iso-seq分析（Iso-seq）获得的cDNA蛋白编码区。最终采用Salmon计算得到的每百万转录本数（TPM，Transcripts per million）对转录本测序读数进行归一化处理。