Phloem and xylem tissue were homogenised in liquid nitrogen with mortar and pestle and then stored at at -80 °C for further RNA or protein extraction. 100 mg of the homogenized tissue was placed in a frozen 2 mL tube containing a ceramic bead and ground for 60 s at a frequency of 26 1/S with a TissueLyser II (Qiagen, Cat. 85,300). Total RNA was isolated using RNeasy Plant Mini kit (Qiagen, Cat. 74,903) with DNase treatments (RNase-Free DNase Set cat. No. 79254) according manufacturer’s instructions. The RNA was quantified using the Qubit 4 fluorometer (ThermoFisher) with the Qubit RNA BR Assay Kit (ThermoFisher, Cat. Q10211). RNA integrity (RIN) was tested with the Agilent RNA 6000 Nano Kit (Agilent, Cat. 5067–1511) on a 2100 Bioanalyzer instrument (Agilent, Cat. G2939BA) and all samples used for RNA-Seq had a RIN greater than 7.
The Illumina NeoPrep Library Prep System was used to prepare samples from 50 ng of total RNA extraction (Illumina, Documents: 15049720v01, 15049725v03, 15059581v02). TruSeq Standard mRNA Library Prep (Illumina, NP-202–1001) was used with the default indexes adapters A to P. At the last step, each processed sample collected from the library card was analysed for library quality check using a DNA 1000 chip on the 2100 Bioanalyzer (Agilent, Cat. 5067–1504). Finally, each sample was normalized manually at 10 nM and then pooled (5 μL × 16 samples) in one library for the Illumina sequencing platform.
The libraries composed of the 16 samples were sequenced into two lines in Rapid-Run Mode (16 samples/line) in a single flow cell for paired-end 100 bp with an Illumina HiSeq 2500 sequencing system. The samples were sequenced at the Centre Hospitalier de l'Université Laval sequencing platform (Quebec City, Canada).
The Illumina NeoPrep Library Prep System was used to prepare samples from 50 ng of total RNA extraction (Illumina, Documents: 15049720v01, 15049725v03, 15059581v02). TruSeq Standard mRNA Library Prep (Illumina, NP-202–1001) was used with the default indexes adapters A to P. At the last step, each processed sample collected from the library card was analysed for library quality check using a DNA 1000 chip on the 2100 Bioanalyzer (Agilent, Cat. 5067–1504). Finally, each sample was normalized manually at 10 nM and then pooled (5 μL × 16 samples) in one library for the Illumina sequencing platform.
The libraries composed of the 16 samples were sequenced into two lines in Rapid-Run Mode (16 samples/line) in a single flow cell for paired-end 100 bp with an Illumina HiSeq 2500 sequencing system. The samples were sequenced at the Centre Hospitalier de l'Université Laval sequencing platform (Quebec City, Canada).
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