Comparative transcriptomic heat stress responses data for native Australian Acacia species: supplementary data and R code

To improve our understanding of heat tolerance across related species from diverse climates, we cultivated a selection of Acacia species in a controlled environment, using seed sourced from wild populations that occupy a wide range of Australian climates. After four months of growth in control temperature conditions (ca 24 °C day and 18 °C night) plants were exposed to a four-day heatwave (38 °C day and 26 °C night). Heatwave responses were quantified with coupled measurements of the transcriptome (gene expression) and PHT acclimation. Samples for transcriptomics were taken across three days: one day prior to heatwave (control), at the onset of heatwave conditions (Day 1) and at the end of the heatwave (Day 4). Accompanying measurements of PHT were taken the day prior and at day four using basal chlorophyll fluorescence.\n\nThis Collections includes RNA-seq metadata and cleaned expression data for data analysis (.rds file) and the associated Rmarkdown file (.Rmd file) to run analyses from our paper.\nLineage: Leaf samples for RNA extraction were taken the morning prior to the heatwave and on the first and fourth morning of the heatwave. Leaf samples were snap frozen with liquid nitrogen before being transported on dry ice to a -80 °C freezer immediately after sampling.\nFor extracting total RNA from Acacia leaf and phyllode tissue, the best method for tissue homogenization proved to be grinding leaf samples with liquid nitrogen in a mortar and pestle. After grinding, samples were returned to dry ice until 16 samples were ready to start the RNA extraction. The kit used for RNA extraction was the NucleoSpin RNA Plant and Fungi Kit (Macherey-Nagel, Germany) using the standard protocol except for an adjustment to the lysis buffer. The lysis buffer aliquot per sample included 400μl of PFL and 50μl PFR buffers from the NucleoSpin Kit, 100μl Fruit-mate for RNA Purification (Takara, Japan) and 5μl of ß-mercaptoethanol. Of the 21 species grown, only 17 species were sequenced at multiple time points. \nAfter mRNA isolation with Oligo d(T)25 Magnetic Beads (New England BioLabs, Australia), strand specific RNA-seq libraries were prepared using an in-house template switching protocol. The protocol for library preps is fulling described in Paten et al., (2022). Two plates of 96 libraries were prepared using custom barcodes. Samples were sequenced on a single NovaSeq S4 flowcell (300 cycles, 2x150bp), using a XP 4-lane splitter kit to split the two sample pools onto 2 lanes each (i.e. set A on lanes 1 and 2 and set B on lanes 3 and 4). Sequencing was performed at the ACRF Biomolecular Resource Facility at John Curtin School of Medical Research at The Australian National University. Funding for sequencing costs was provided by Bioplatforms Australia.\n\nA full list of libraries and associated meta-data is provided in “Acacia_metadata_DAP_220523.xlsx” (see link = https://data.csiro.au/collection/csiro:57771). RNA-seq libraries are uploaded in two batches because of file sizes (part 1 = https://data.csiro.au/collection/csiro:57772 and part 2 = https://data.csiro.au/collection/csiro:57773). \n\nA. M. Paten, et al., Non-additive gene interactions underpin molecular and phenotypic responses in honey bee larvae exposed to imidacloprid and thymol. Sci. Total Environ. 814 (2022).\n

为加深对不同气候下近缘物种耐热性的认知，我们在可控环境下栽培了一组金合欢属（Acacia）物种，所用种子采自分布于澳大利亚多种气候区域的野生种群。
在对照温度条件（日间约24℃、夜间18℃）下生长四个月后，将植株置于为期四天的热浪环境（日间38℃、夜间26℃）中进行处理。通过转录组（transcriptome，即基因表达）与PHT驯化的联合检测，量化植株的热浪响应。转录组学样本采集于三个时间点：热浪处理前1天（对照）、热浪处理启动当日（第1天）以及热浪处理结束当日（第4天）。PHT的配套检测则于热浪处理前1天及第4天，通过基础叶绿素荧光完成。

本数据集包含用于数据分析的RNA测序（RNA-seq）元数据与标准化表达数据（.rds格式文件），以及可复现本文分析流程的关联Rmarkdown文件（.Rmd格式文件）。

样本采集流程：用于RNA提取的叶片样本采集于热浪处理前一日清晨，以及热浪处理第1、4日的清晨。样本采集后立即用液氮快速冷冻，随后置于干冰中运输，最终保存于-80℃冰箱。针对金合欢属叶片与叶状柄组织的总RNA提取，经验证最优的组织匀浆方法为使用研钵和研杵结合液氮对叶片样本进行研磨。研磨完成后，样本放回干冰保存，直至集齐16份样本启动RNA提取流程。本次RNA提取采用NucleoSpin RNA Plant and Fungi Kit（德国Macherey-Nagel公司），严格遵循标准操作流程，仅对裂解缓冲液进行了调整。每份样本的裂解缓冲液组分如下：NucleoSpin试剂盒自带的PFL缓冲液400μl、PFR缓冲液50μl，RNA纯化用Fruit-mate试剂（日本Takara公司）100μl，以及β-巯基乙醇5μl。本次栽培的21个金合欢属物种中，仅17个物种完成了多时间点的测序。

使用Oligo d(T)25磁珠（澳大利亚New England BioLabs公司）完成mRNA富集后，采用自主开发的模板转换法制备链特异性RNA测序文库。文库制备的详细流程已在Paten等人2022年的研究中完整阐述。本次共制备2块96孔板的定制条形码文库。测序在单张NovaSeq S4流动槽上完成（300个循环，2×150bp读长），使用XP 4泳道分流试剂盒将两个样本池分别分配至2个泳道（即样本组A分配至泳道1、2，样本组B分配至泳道3、4）。测序工作由澳大利亚国立大学约翰·科廷医学研究院的ACRF生物分子资源设施完成，测序成本由Bioplatforms Australia资助。

完整的文库及关联元数据列表已上传至"Acacia_metadata_DAP_220523.xlsx"（链接：https://data.csiro.au/collection/csiro:57771）。由于文件体积较大，RNA测序文库分为两批上传（批次1：https://data.csiro.au/collection/csiro:57772；批次2：https://data.csiro.au/collection/csiro:57773）。

A. M. Paten等，《非加性基因互作支撑暴露于吡虫啉与百里酚的蜜蜂幼虫分子与表型响应》，《Science of the Total Environment》，814卷（2022年）。