Transcript profiles of developing Arabidopsis dgat1-1 mutant seed

Global transcript profiling of Arabidopsis dgat1-1 seed at different stages of embryo development was used to identify differentially expressed genes in the mutant compared to wild-type (Col-0) seed. These genes provided information about the remodeling of lipid metabolism and TAG synthesis in response to the lack of DGAT1 activity. In depth transcriptome profile was generated using RNA-Seq for total RNA extracted from different seed development stages 8, 12, 16 Days after flowering (DAF). Samples were collected from wild type (WT) and dgat1-1 genotypes, with three biological replicates for each time point/genotype combination. RNA was sequenced at the Genomics Core at the University of Kansas Medical Center. The mRNA fraction was enriched with oligo dT capture, sized, reverse transcribed into cDNA and ligated with the appropriate indexed adaptors using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina). Following Agilent Bioanalyzer QC of the library preparation and library quantification using the Roche Lightcycler96 with KAPA SYBR Fast Universal qPCR kit (KAPA Biosystems), the RNA-Seq libraries were adjusted to a 4nM concentration and pooled for multiplexed sequencing. Libraries were denatured and based on qPCR results, diluted to the appropriate concentration, followed by clonal clustering onto the sequencing flow cell using the TruSeq Paired-End (PE) Cluster Kit v3-cBot-HS (Illumina). The clonal clustering procedure was automated using the Illumina cBOT Cluster Station. The clustered flow cell was sequenced on an Illumina HiSeq 2500 Sequencing System using the TruSeq SBS Kit v3-HS (Illumina) to obtain 100bp pair-end reads, which were trimmed and quality assessed before assembly against the Arabidopsis reference genome (Araport 11) using CLC genomics workbench (v 7.5.1)本研究对胚胎发育不同阶段的拟南芥dgat1-1突变体种子开展全局转录组分析，以鉴定该突变体相较于野生型（Col-0）种子的差异表达基因，这些基因可为解析DGAT1活性缺失时脂质代谢重塑及三酰甘油（TAG）合成的调控机制提供依据。本研究针对开花后8、12、16天（DAF）的不同种子发育阶段提取的总RNA，通过RNA测序（RNA-Seq）构建了深度转录组图谱；实验样本取自野生型（WT）及dgat1-1两种基因型，每个时间点-基因型组合均设置3次生物学重复。测序工作于堪萨斯大学医学中心基因组学核心实验室完成，实验采用寡聚dT捕获法富集mRNA片段，经片段大小筛选后，使用TruSeq链特异性mRNA样本制备试剂盒（Illumina）反转录合成cDNA，并连接适配的带索引接头；文库制备完成后，通过安捷伦生物分析仪（Agilent Bioanalyzer）进行质量控制，并采用罗氏Lightcycler96实时荧光定量PCR仪结合KAPA SYBR Fast通用qPCR试剂盒（KAPA Biosystems）完成文库定量，随后将RNA测序文库调整至4nM浓度并混合以用于多重测序。将文库变性后，基于qPCR定量结果稀释至适宜浓度，随后使用TruSeq双端（Paired-End，PE）簇生成试剂盒v3-cBot-HS（Illumina）在测序流动槽上完成克隆簇扩增，该流程通过Illumina cBOT簇生成工作站实现自动化操作；最终使用TruSeq SBS试剂盒v3-HS（Illumina）在Illumina HiSeq 2500测序系统上对携带克隆簇的流动槽进行测序，获取100bp双端读长序列，后续通过CLC基因组工作台（v7.5.1）对原始序列进行修剪与质量评估，再与拟南芥参考基因组（Araport 11）进行比对组装。