RNA extraction for qRT-PCR and RNAseq

Top shoot tip leaves (~ 50 mg) were collected in Precellys lysing tubes, flash frozen with liquid nitrogen, homogenized by a PowerLyzer 24 and total RNA was extracted using QIAGEN RNeasy Plant mini kit. Total RNA was treated with Ambion TURBO DNA-free DNase followed by iScript cDNA synthesis for qRT-PCR using the CFX96 Real-Time PCR detection system (Bio-Rad). Specifically, we mixed 2 µL(200 ng) cDNA, 1 µL forward and reverse gene-specific primers (10 µM each) (Table ^S1), 5 µL SsoFast EvaGreen Supermixes, and 2 µL of nuclease-free water. PCR amplification was performed using: cDNA denaturation at 95 °C for 30 s followed by 40 cycles of 95 °C for 10 s, 58 °C for 30 s and 72 °C for 30 s followed by a melting curve that ran from 65 to 95 °C with a gradual increment of 0.5 °C per 5 s. All reactions were performed with three technical and four biological replicates. Transcript levels were analysed relative to acetyl-CoA carboxylase (ACC1) and ACTIN housekeeping genes.

Total RNA quality was checked using a BioRad Bioanalyzer before RNAseq analysis. Stranded mRNA library was prepared with NEBNext followed by sequencing with Illumina NovaSeq6000 with 100 bp fragment pair end reads at Genome Quebec, Montreal as a fee-for-service. Raw RNA sequencing reads can be accessed from the National Center for Biotechnology Information, NCBI, BioProject PRJNA596791.