RNA-seq Library Preparation and Sequencing

RNA-seq library was prepared from RNA isolated from three biological replicates of phyB-1 and wild-type axillary buds at 6, 7, and 8 DAP. Total RNA was isolated from at least six axillary buds per sample using the TRIzol method (Invitrogen, Carlsbad, CA). RNA-seq libraries were prepared using the TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA), following the recommended protocol. Briefly, mRNA was purified from 400-ng total RNA using poly-T oligo-attached magnetic beads. The mRNA was eluted, fragmented, and primed for cDNA synthesis. After first- and second-strand synthesis, the cDNA was end-repaired, adenylated, and unique adapters were ligated to each sample and the DNA fragments were amplified before sequencing. The quality of each RNA-seq library was assessed using a bioanalyzer and sequenced using HiSEquation 2500 (Illumina) at the TX A&M Genomics and Bioinformatics Services Center (College Station, TX).