Study : Transcript profiles of developing Arabidopsis dgat1-1 mutant seed


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. Overall design: 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)


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