Agilent 2100 bioanalyzer high sensitivity or dna 1000 assay

Total RNA was extracted from homogenized samples of developing xylem of four biological replicates per genotype and treatment using the CTAB protocol (Chang et al. 1993 ) with minor modifications described in Janz et al. (2012) (link). Isolated RNA was quality checked using Agilent 2100 Bioanalyzer RNA Nano assay (Agilent Technologies, Santa Clara, CA, USA). Two micrograms of total RNA (RNA Integrity Number RIN >7.0) was used for library preparation using the ‘TruSeq mRNA Sample Prep kit v2’ (Illumina, San Diego, CA, USA), following the manufacturers’ instructions. Final libraries were quantified using the Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and quality tested by Agilent 2100 Bioanalyzer High Sensitivity or DNA 1000 assay (Agilent Technologies). Successively, libraries were loaded on Illumina cBot for cluster generation on the flow cell, following the manufacturer’s instructions and sequenced in 50 bp single-end mode at sixfold multiplex on the Illumina HiSeq2000 (Illumina). The CASAVA 1.8.2 version of the Illumina pipeline was used to process raw data for both format conversion and de-multiplexing. In average, 26.91 million reads were produced per sample (min 21.12). Short reads have been deposited into NCBI Short Read Archive under BioProject accession PRJNA359401.