Transcriptome analysis of RGA1 mutant (d1-1) meristem by RNA-seq

We applied RNA-seq to 3-month-old rice (Oryza sativa) shoot apical meristem to investigate the effect of RGA1(d1-1) mutation on the meristem initiation using transcriptome of d1 as compared to wild type plant. Overall design: Two biological replicates of rice shoot apical meristem were dissected out from leaf sheath and then collected from three-month-old WT and d1 plants grown as described above and immediately frozen in liquid nitrogen. Total RNA was extracted from each sample using the NucleoSpin RNA Plant kit. After examination of the quality (Bioanalyzer, Agilent) and quantity (NanoDrop 2000, ThermoFisher) of each RNA sample, aliquots of total RNA were submitted to the Genomics Core Facility at Penn State University for cDNA library preparation and next generation sequencing on a Hiseq 2500 (Illumina). Approximately 40 million 150 bp single-end sequencing reads were obtained for each library.

本研究采用RNA测序（RNA-seq）技术对3月龄水稻（Oryza sativa）的茎尖分生组织进行转录组测序，以野生型（Wild Type, WT）植株为对照，通过对比d1突变体的转录组数据，探究RGA1(d1-1)突变对分生组织起始过程的影响。
实验整体设计如下：从上述培养的3月龄野生型及d1突变体水稻植株的叶鞘中剥离茎尖分生组织，设置两个生物学重复样本，采集后立即置于液氮中速冻保存。采用NucleoSpin RNA Plant试剂盒提取各样本的总RNA；经生物分析仪（Bioanalyzer, Agilent）检测RNA样本质量、NanoDrop 2000（ThermoFisher）检测RNA样本浓度后，取适量总RNA送至宾夕法尼亚州立大学基因组核心实验室开展cDNA文库构建，并在Illumina HiSeq 2500测序平台上完成下一代测序。每个文库均可获得约4000万条150bp的单端测序读段。