Agilent tapestation 1500

Samples treated with two ANE doses were harvested 24h and 48h after treatment for RNA-Seq analysis together with controls.
Two leaf disks were collected around the mid-vein of the distal leaflets of the most recently fully expanded leaf below the nearest inflorescence, from four different plants for each experimental condition. Messenger RNA was directly isolated from frozen and powdered leaf disk pools using the Dynabeads mRNA Direct Micro Kit (Thermo Fisher Scientific, Carlsbad, CA) following the manufacturer’s instruction. The concentration and quality of mRNA were assessed by an Agilent 4150 TapeStation system (Agilent Technologies, USA). Sequencing libraries were prepared from a range of 10-50 ng of poly(A) RNA using Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific) following the manufacturer’s protocol. The final double-stranded barcoded cDNA libraries were eluted in 15 µl of nuclease-free water. The concentration and size distribution were quantified through D1000 screen Tape (Agilent Tapestation 1500), normalized to get a molar concentration of 100pM, pooled, and sequenced using three Ion 540™ Chips on the Ion Torrent S5 System (Thermo Fisher Scientific).

A total of 30 mg of fresh leaf samples were processed using Tissue Lyser (Qiagen, Germany) with 100 µL of the lysis-binding buffer. mRNAs were directly isolated using the Dynabeads mRNA Direct Micro Kit (Thermo Fisher Scientific, Carlsbad, CA, USA). The quality and quantity of isolated mRNAs were checked by Agilent TapeStation 1500 (Agilent Technologies Inc., Santa Clara, CA, USA). Sequencing libraries were prepared using Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific). The prepared libraries were loaded onto Ion 540 chip kit followed by sequencing using the Ion S5 GS system.