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Protocol publication

[…] Total RNA from fresh and 4 day-old cold-stored Asparagus spears was extracted using the Isolate Plant RNA Mini Kit (Bioline, London, UK) and treated with DNase I (Invitrogen, Carlsbad, California, US) according to the manufacturer’s instructions to remove genomic DNA contamination. Total RNA samples were then quantified using a Nano-Drop (Thermo Scientific, Rockford, IL, US) and 10 μg total RNA submitted to AGRF (Melbourne, Victoria, Australia) for library construction (without size selection) and sequencing on a HiSeq 2000 (Illumina). Raw RNA-seq data from a single lane (191,179,245 read paired-end reads) was subjected to a quality control step involving adaptor removal and sequence trimming using Nesoni (http://www.vicbioinformatics.com/software.nesoni.shtml, min. length 30, quality 24, first 10 bp trimmed) and then de novo-assembled using Trinity []. The assembly was then scaffolded using CAP3 [] (default settings) for each Trinity component.A quantitative RNA-Seq experiment was also conducted using four biological replicates of apical, middle and basal section from fresh Asparagus spears, each replicate comprising tissue sections from five separate spears. Samples were run on an Illumina HiSeq 2000 in two lanes in a single flow-cell. All samples were sequenced in both lanes. Reads from each sample from each lane were merged and then filtered for quality and adaptor removal as per the reference sample. Reads for each sample were then quantitated independently against the reference transcriptome using RSEM v1.2.8 []. Orphaned reads were not used for quantitation. EBSeq v1.1 [] was used to identify differentially expressed genes.Candidate xylan biosynthetic genes and several known secondary cell wall regulation genes were identified in the Asparagus reference transcriptome by BLASTX searches (NCBI BLAST+ v2.2.25) using Arabidopsis sequences as bait. [...] Sequence alignments and phylogenetic analysis was performed using Clustal X 2.1 []. IRX proteins from Arabidopsis thaliana (At), Asparagus officinalis (Ao), rice (Oryza sativa, Os) and moss (Physcomitrella patens, Pp) were aligned using Clustal X 2.1 and used as input to produce a neighbor-joining tree with 1000 bootstrap replicates using default settings. […]

Pipeline specifications

Software tools Nesoni, Trinity, CAP3, RSEM, EBSeq, BLASTX, Clustal W
Applications Phylogenetics, RNA-seq analysis
Organisms Arabidopsis thaliana