RNA-seq on tomato genotypes exposed to white and white plus far red light: IL2-2 genotype treated with white plus far red light replicate 1
Total RNA was extracted from leaves at seedling stage of M82 and the introgression line IL2.2 under white and white plus far red conditions. RNA extraction was carried out using the ISOLATE II RNA Plant Kit (Meridian Bioscience, Cincinnati, Ohio, USA ) according to the method reported by the manufacturer. Three independent replicates of total RNA (1 g) extracted from leaves of different plants growing under the same conditions was used for RNA sequencing. mRNA enrichment was performed on total RNA using oligo(dT)-attached magnetic beads. The enriched mRNA with poly(A) tails was fragmented using a fragmentation buffer, followed by reverse  transcription using random N6 primers to synthesize cDNA double strands. The synthesized double stranded DNA was then end-repaired and 5'-phosphorylated, with a protruding 'A' at the 3' end  forming a blunt end, followed by ligation of a bubble-shaped adapter with a protruding 'T' at the 3' end. The ligation products were PCR amplified using specific primers. The PCR products were denatured to single strands, and then single-stranded circular DNA libraries were generated using a bridged primer. The constructed libraries were quality-checked and sequenced after passing the quality control. Sequencing was performed on DNBSEQ platform with PE150 (read length).