RNA-Seq of Arabidopsis thaliana: bar gene GM under control conditions (Replicate 2)
RNA was extracted using the TRIzol method (Invitrogen, CA, USA) and treated with RNase-free DNase I (Takara, Kusatsu, Japan). RNA quality was assessed using 1% agarose gel electrophoresis, NanoDrop spectrophotometer (Thermo Scientific, DE, USA), and Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). A total of 1.5 g RNA per sample was used for library construction. mRNA was purified using poly-T oligo-attached magnetic beads. Fragmentation was performed under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First-strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase. Second-strand cDNA synthesis was performed using DNA Polymerase I and RNase H. cDNA fragments were purified using AMPure XP beads and size-selected to 200-250 bp. Libraries were constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA), with adapters added. Sequencing was performed on an Illumina NovaSeq 6000 platform to generate 150 bp paired-end reads.