title: RNA-Seq of Arabidopsis thaliana: lox2-U1-3
design: Total intact RNA extractions were performed from flash-frozen pulverized plant rosettes (~100 mg) using the RNeasy Plant Mini Kit (Qiagen) following the manufacturers protocol with an additional on-column DNase digestion step to avoid genomic DNA contamination. The 2100 Bioanalyzer instrument (Agilent Technologies) was used to determine RNA quality using an RNA integrity number (RIN) of 8.7 as a minimum threshold. Total RNA samples were processed by Genome Qubec Innovation Centre for library preparation and next-generation RNA sequencing (RNA-Seq). Libraries were generated from 250 ng of total RNA as follows: mRNA enrichment and ribosomal RNA removal was performed using the Next Poly(A) Magnetic Isolation following the manufacturers instructions (New England BioLabs). cDNA synthesis was performed with Next RNA First Strand Synthesis and Next Ultra Directional RNA Second Strand Synthesis kits (New England BioLabs). The remaining steps of library preparation were performed using the Next Ultra II DNA Library Prep Kit for Illumina (New England BioLabs). Adapters and PCR primers were purchased from New England BioLabs. The libraries were normalized, pooled and denatured in 0.02 N NaOH, followed by neutralization using HT1 hybridization buffer. Twenty-four strand-specific mRNA libraries were generated and sequenced by loading 200 pM on the NovaSeq 6000 S4 (Illumina) using the Xp protocol as per the manufacturers recommendations, with paired end (PE) mode for 2 x 100 base pairs (bp) resulting in 25 million reads in each direction. The Illumina phiX control v3 library was used as a control and mixed with libraries at a 1% level. Base calling was performed using Real Time Analysis (RTA) v3 software. The program bcl2fastq2 v2.20 was used to demultiplex samples and generate fastq reads.
library_name: lox2-U1-3