RNA-Seq of Pinus torreyana: twig tissue
We extracted total RNA from twig tissue using the Spectrum Plant Total RNA Kit (Sigma-Aldrich).  For twigs, we used a razor blade to remove the outer bark layer to reduce lichen and other potential contaminants.  We verified RNA quality and quantity using a Nanodrop One spectrophotometer (Thermo Scientific), Qubit BR Assay kit (Life Technologies) and an Agilent 2100 Bioanalyzer, and cleaned extracts with AMPure XP beads as needed.  We prepared mRNA libraries with a TruSeq Stranded mRNA Kit (Illumina) and unique dual index adapters.  Libraries were PCR amplified for 15 cycles, quantified by Qubit, and validated on an Agilent 2100 Bioanalyzer.  We performed double size selection with AMPure XP beads as needed to reduce minor secondary peaks in the Bioanalyzer trace.  We sequenced final libraries as paired end 75 bp reads across four runs on an Illumina NextSeq500 at Sanford Burnham Prebys Discovery Institute.