Transcriptomic analysis revealed adverse effect of melatonin on root growth. Transcriptomic analysis revealed adverse effect of melatonin on root growth

Purpose: We aimed to compare transcriptomic changes after high concentration melatonin treatment in Arabidopsis Methods: A total amount of 3 μg RNA was used for generation of sequencing libraries using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated. Clean reads were obtained by removing low quality reads, reads containing adapter and ploy-N from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. Index of the Arabidopsis genome was built using Bowtie v2.2.3 and paired-end clean reads were aligned to the reference genome using TopHat v2.0.12. HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. And then FPKM (Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced) of each gene was calculated based on the length of the gene and reads count mapped to this gene. Differential expression analysis of drought stress versus control condition was performed using the DESeq R package (1.18.0). Results:In total, eight samples with two biological replicates per genotype/treatment combination were used for RNA sequencing analysis. At least 2 G clean bases were generated for each sample. Comparative analysis revealed different networks which were modulated by melatonin and IAA in Arabidopsis seedlings Overall design: In this study, wild type Arabidopsis plants were grown in MS plate for 7 days.Plants were then treated with 0,100 and 1000μM melatonin for 12h. The rosette leaves were then collected for RNA isolation and deep sequencing.

研究目的：本研究旨在比较高浓度褪黑素处理后拟南芥（Arabidopsis）的转录组变化。
方法：取3 μg总RNA，参照厂商推荐操作流程，使用NEBNext® Ultra™ 适配Illumina®平台RNA文库制备试剂盒（美国纽英伦生物技术有限公司，NEB）构建测序文库，并添加索引标签以将序列归属至对应样本。完成簇生成后，在Illumina HiSeq平台对文库进行测序，获得125 bp/150 bp双端读段。通过从原始数据中剔除低质量读段、含接头读段及ploy-N读段，获得洁净读段；同时计算洁净数据的Q20、Q30值与GC含量。使用Bowtie v2.2.3构建拟南芥基因组索引，再通过TopHat v2.0.12将双端洁净读段比对至参考基因组。使用HTSeq v0.6.1统计比对至每个基因的读段数量；随后基于基因长度及比对至该基因的读段数，计算每个基因的FPKM（每百万测序碱基片段对应每千碱基转录本片段数，Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced）。使用DESeq R包（1.18.0）开展干旱胁迫组与对照组的差异表达分析。
结果：本研究共纳入8个样本，每个基因型/处理组合设置2个生物学重复，用于RNA测序分析。每个样本至少生成2 G洁净碱基。比较分析显示，褪黑素与吲哚-3-乙酸（IAA）可调控拟南芥幼苗的多条信号通路网络。
实验设计：本研究中，野生型拟南芥植株在MS培养基平板上培养7天；随后分别用0、100及1000 μM褪黑素处理12小时，收集莲座叶用于RNA提取与深度测序。