Computational protocol: Glyphosate’s impact on vegetative growth in leafy spurge identifies molecular processes and hormone cross-talk associated with increased branching

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Protocol publication

[…] Total RNA was purified from the eight aerial tissue samples derived from crown buds of foliar glyphosate-treated plants according to the pine tree RNA extraction protocol []. The RNA was treated with amplification grade DNase1 (Invitrogen), quantified by the Qubit® 2.0 Fluorometer, and quality was confirmed by agarose gel electrophoresis. The RNAseq libraries were prepared with the TruSeq Stranded mRNA LT Sample Preparation Kit (Illumina, Cat. N° RS–122–2101) starting from 1 μg of total RNA. The pool of barcoded RNAseq libraries was quantified by qRT-PCR using the Library Quantification kit (Kapa Biosystems, Cat. N° KK4824). The size range of the final cDNA libraries was determined on an Agilent bioanalyzer DNA7500 DNA chip (Agilent Technologies). The cDNA libraries were sequenced on one lane for 151 cycles from each end of the cDNA fragments on a HiSeq2500 using a TruSeq SBS sequencing kit version 1 (Illumina). The sequence images were transformed with the Real Time Analysis 1.17.21.2 Illumina software to bcl files, which were demultiplexed to fastq files with CASAVA version 1.8.2. The quality-scores line in fastq files processed with Casava1.8.2 use an ASCII offset of 33 known as Sanger scores. [...] The Trinity package version trinityrnaseq-r2013-02-25 [] was utilized to assemble the transcriptome from the RNASeq data. Prior to assembly, adaptor sequences were trimmed and so were any low quality bases (<30 phred score) from both ends of the sequence. Sequences of 50 nucleotide and higher were utilized for the Trinity assembly. The assembly was performed as per guidelines in the published protocols [], and on the Trinity website (http://trinityrnaseq.sourceforge.net/#running_trinity). The abundance of every gene (component) in each of the eight samples was determined by performing alignment and abundance estimation using the RSEM package [] (version 1.2.3). Only those genes that had at least one sample with the transcripts per million (TPM) value of ≥ 0.5 were used as input for the next step. Differential gene expression was performed at the level of genes with the EBSeq package [] (version 1.0) using the median normalization approach and 10 iterations of the algorithm used by EBSeq. Genes were called differentially expressed if they had an FDR value of ≤0.05.Annotation of the assembled transcriptome and predicted proteome was performed with the Trinotate package (version trinotate_r20130826). Transdecoder, a Perl script packaged with Trinity, was used to predict proteins, yielding the most likely longest-ORF peptide candidates file best_candidates.eclipsed_orfs_removed.pep. Analyses were performed as detailed in the Trinotate manual (http://trinotate.sourceforge.net/), with the following noted exceptions. BLASTx of assembled transcripts and BLASTp most likely longest-ORF peptide candidates against UniProt-SwissProt database were run with the options “-evalue 10E-3 –max_target_seqs 5” []. For more reliable functional annotation, BLASTx of Trinity.fasta and BLASTp of best_candidates.eclipsed_orfs_removed.pep against Arabidopsis peptides were performed (Athaliana_167_protein.fa from Phytozome v9.0). BLAST output files were parsed to 1 top hit per query with an in-house Perl script prior to insertion in the Trinotate SQLite database (http://trinotate.sourceforge.net/#LoadingSQLite). After extraction of the Trinotate annotation report from the database (http://trinotate.sourceforge.net/#OutputReport), Arabidopsis annotations were added using an in-house Perl script. The expression data is deposited at Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) as GEO dataset query GSE56509. […]

Pipeline specifications

Software tools BaseSpace, Trinity, RSEM, EBSeq, Trinotate, TransDecoder, BLASTX, BLASTP
Applications RNA-seq analysis, Transcription analysis
Chemicals Abscisic Acid, Cytokinins, Gibberellins