Agilent 2100 bioanalyser plant nano system

RNA integrity was confirmed using the Agilent 2100 Bioanalyser Plant Nano system (Agilent Biotechnologies). Stranded Illumina TruSeq libraries were generated from 1 μg of total RNA and sequenced (100 bp paired end reads) on an Illumina HiSeq platform by the Australian Genome Research Facility (AGRF). 51–60 million reads were generated for each sample. RNA-seq paired-end reads were trimmed for low-quality base-calls and Illumina adapter sequences via Cutadapt (v1.1, parameters: −quality-cutoff 30 --overlap 10 --times 3 –minimum-length 25) [93 (link)]. Reads trimmed to less than 25 bp were discarded and remaining reads sorted into pairs and singleton reads. RNA-seq reads were mapped to the Fom-5190a genome assembly via Tophat2 (v2.0.9, parameters: −b2-very-sensitive -r 80 --mate-std-dev 40 -i 20 -I 4000 -g 20 --report-secondary-alignments --report-discordant-pair-alignments -m 0 --min-coverage-intron 20 --microexon-search --library-type fr-firststrand) [140 (link)].

RNA integrity was confirmed using the Agilent 2100 Bioanalyser Plant Nano system (Agilent Biotechnologies). Stranded Illumina TruSeq libraries were generated from 1 μg of total RNA and sequenced (100 bp paired end reads) on an Illumina HiSeq platform by the Australian Genome Research Facility (AGRF). As per [5 (link)], RNA-Seq paired-end reads were trimmed for low-quality base-calls (≥ q30) and Illumina adapter sequences using Cutadapt v1.1 [84 ] (parameters: --quality-cutoff 30 --overlap 10 --times 3 –minimum-length 25) with reads trimmed to less than 25 bp discarded and remaining reads sorted into pairs and singleton reads. RNA-Seq reads were then mapped to the Fom-5190a genome assembly via Tophat2 (TopHat2 v2.0.9) (parameters: --b2-very-sensitive -r 80 --mate-std-dev 40 -i 20 -I 4000 -g 20 --report-secondary-alignments --report-discordant-pair-alignments -m 0 --min-coverage-intron 20 --microexon-search --library-type fr-firststrand) [30 (link)]. BAM files generated from Tophat2 output were sorted using SAMtools version 0.1.19 [85 (link)] and visualised using IGV (version 2.3) [86 (link)]. Trimmed sequencing data is available from the NCBI/GenBank database under BioProject number PRJNA294248 (http://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA294248).