Next Generation Sequencing to Study FLK-affected genes. Next Generation Sequencing to Study FLK-affected genes

Purpose: The goals of this study are to compare RNA-seq profiles of Col-0, acd6-1, flk-1, flk-5, acd6-1flk-1, and acd6-1flk-5 to identify genes affected by FLK. Methods: Total RNA was extracted from 25-d old plants. A total amount of 1 μg RNA per sample was used to generate cDNA libraries, using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. Deep sequencing was performed using Illumina NovaSeq 6000 (Novogene Corporation Inc.). Triplicate biological samples were used for all genotypes except acd6-1flk-5, which had duplicate samples because one sample failed to pass quality control. The samples were multiplexed and sequenced with the standard paired-end sequencing that has a read length of 150bp per end and 20M reads per end per sample. Results: We found that over 8000 genes are differentially affected by FLK based on the RNAseq analysis. GO analysis revealed that FLK-affected genes are enchriched for genes involved in primary metabolism and in response to abiotic and biotic stimuli. Conclusions: The RNAseq analysis support a role of FLK in regulation of plant development and defense. Overall design: 25-d old plants were collected for RNA extraction followed by cDNA library preparation and high throughput.

研究目的：本研究旨在比对Col-0、acd6-1、flk-1、flk-5、acd6-1flk-1及acd6-1flk-5的RNA测序（RNA-seq）表达谱，以鉴定受FLK调控的靶基因。
方法：从生长25天的植株中提取总RNA。每个样品取1 μg总RNA，参照美国纽英伦生物（NEB, USA）的NEBNext® UltraTM RNA文库制备试剂盒（适配Illumina®平台）的厂商操作指南构建cDNA文库。采用Illumina NovaSeq 6000平台由诺禾致源公司（Novogene Corporation Inc.）完成深度测序。除acd6-1flk-5基因型仅设置2个生物学重复（其中1个样品未通过质量控制）外，其余所有基因型均设置3次生物学重复。所有样品采用多重测序策略，使用标准双端测序模式，每端读长为150 bp，每个样品每端产出20M条读段。
结果：通过RNA-seq分析发现，超过8000个基因的表达受FLK调控呈现显著差异。基因本体（Gene Ontology）分析显示，受FLK调控的基因显著富集于初级代谢过程以及应对非生物和生物胁迫的通路中。
结论：RNA测序分析证实FLK在植物生长发育与防御反应调控中发挥关键作用。
整体实验设计：收集生长25天的植株进行RNA提取，随后构建cDNA文库并开展高通量测序。