Next Generation Sequencing of Sorted pWER::GFP Marker Line Enables Quantitative Analysis of Arabidopsis Epidermal Root Transcriptomes in Iron Deficient Conditions. Next Generation Sequencing of Sorted pWER::GFP Marker Line Enables Quantitative Analysis of Arabidopsis Epidermal Root Transcriptomes in Iron Deficient Conditions

Purpose: Identify novel genes and relationships of a temporal transcriptional landscape in Arabidopsis epidermal root cells under iron deficiency stress Results: We identified 1,709 novel iron responsive genes which would have been missed had we identified DEGs with respect to the 0hr time point using the whole root or epidermis data. Conclusion: Gene set comparisons allowed us to identify organ-level response versus cell-specific and to parse out developmental from iron deficiency-induced response. We identified several new iron deficiency-induced and repressed TFs and implemented a novel sampling method to generate an epidermis specific GRN that incorporates gene-level expression while maintaining computational efficiency. Using cis-elements in the promoters of DEGs, we were also able to find plausible regulators that would not have been found by exclusively looking at differentially expressed genes. The new transcriptional data provides clarity to our current understanding of root responses to -Fe triggered by the first layer of “defense,” the root epidermis. Overall design: RNA-Seq was performed on protoplast collected from GFP sorted epidermis marker line (pWER::GFP; WEREWOLF; AT5G14750; Lee & Schiefelbein, 1999). Arabidopsis roots were collected on the 7th day of growth on +Fe media, equating to 0hr. After 7days, seedlings were transfer to +Fe and -Fe media, an additional 6hr, 12hr, 18hr, 24hr, 30hr, and 36hr of growth occurred. After which, roots were harvested from both +Fe and -Fe conditions for protoplast collection, followed by fluorescence-activated cell sorting in triplicates. RNA was extracted from FACS epidermal protoplast using RNAeasy Plant RNA Purification Kit (Qiagen). cDNA synthesis and amplification were performed using the SMARTer Low Input RNA Kit and sequenced using an Illumina HiSeq 2500 sequencing machine, with 125bp single end reads. Adapters and low quality reads were filtered out using fastQC and fastq-mcf. Clean reads were mapped against the TAIR 10 reference genome using the tophat2. RPKM were acquired using RSubread, followed by edgeR to identify differentially expressed genes (DEGs), using a maximum false discovery rate (FDR) of 0.05 and minimum log fold change threshold of 0.75.

研究目的：鉴定缺铁胁迫下拟南芥根表皮细胞时序转录调控景观中的新型基因及其关联关系。
研究结果：本研究共鉴定出1709个新型铁响应基因；若仅以全根或表皮组织的0小时时间点作为参照鉴定差异表达基因（Differentially Expressed Genes, DEGs），则会遗漏这批基因。
研究结论：通过基因集比较分析，我们得以区分器官水平响应与细胞特异性响应，并解析出发育相关响应与缺铁诱导响应。本研究鉴定出多个新的缺铁诱导型及抑制型转录因子（Transcription Factors, TFs），并开发了一种新型采样方法，用于构建整合基因表达水平信息且保持计算效率的表皮特异性基因调控网络（Gene Regulatory Network, GRN）。借助差异表达基因启动子区域的顺式作用元件，我们还发现了仅通过差异表达基因分析无法找到的潜在调控因子。本研究新增的转录组数据，为我们理解由根表皮这一植物抵御缺铁胁迫的第一道"防线"所介导的根系响应机制提供了更清晰的认知。
实验设计：本研究以表达绿色荧光蛋白（Green Fluorescent Protein, GFP）的根表皮标记株系（pWER::GFP; WEREWOLF; AT5G14750; Lee & Schiefelbein, 1999）的原生质体为材料开展RNA测序。将拟南芥幼苗在含铁（+Fe）培养基上培养7天，此时记为0小时时间点。随后将幼苗转移至含铁（+Fe）与不含铁（-Fe）培养基中，分别继续培养6、12、18、24、30及36小时。在各时间点分别收取+Fe与-Fe条件下的幼苗根系，分离原生质体后通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）进行三次生物学重复取样。采用RNeasy植物总RNA提取试剂盒（Qiagen）从分选得到的表皮原生质体中提取RNA。使用SMARTer低起始量RNA试剂盒完成cDNA合成与扩增，随后利用Illumina HiSeq 2500测序平台进行125bp单端测序。使用fastQC与fastq-mcf过滤接头序列及低质量读段。将过滤后的清洁读段比对至TAIR 10参考基因组，比对工具为tophat2。采用RSubread计算每百万比对reads每千碱基转录本的片段数（Reads Per Kilobase per Million mapped reads, RPKM），随后通过edgeR包鉴定差异表达基因，筛选阈值为最大错误发现率（False Discovery Rate, FDR）≤0.05，最小对数倍变化阈值为0.75。