title: RNA-seq of Juglans regia: pellicle
design: RNA was extracted with Invitrogen PureLink Plant RNA Reagent, purified using Qiagen RNeasy MinElute RNA Cleanup Kit and Qiagen RNAse-Free DNAse. Library prepared using Illumina TruSeq Stranded mRNA Library Prep Kit: mRNA was purified using poly-T magnetic beads; mRNA was fragmented using divalent cations, 94C, 8 min. Cleaved mRNAs were copied into first strand cDNA using reverse transcriptase and random primers followed by second strand synthesis. The 3 end of each cDNA fragment was adenylated and sample-specificBIOO Scientific NEXTflex Rapid Directional RNA-Seq adapter sequences were ligated to the cDNA fragments prior to PCR amplification. cDNA concentration was determined using a Quibit 3.0 fluorometer with High Sensitivity assay kit; quantified libraries were diluted to a standard concentration for pooling and clustering. Library integrity was evaluated with an Agilent DNA 1000 chip on an Agilent 2100 bioanalyzer. Indexed libraries were normalized to 10 nM; 10 uL of each library transferred to new PCR plate. Quantitative real-time PCR performed using cluster-forming fragments to generate consistent cluster counts. A KAPA library quantification kit was used for quantification of the number of adapter-ligated molecules present in each library to enable equal loading of the flow cell. An Illumina cBot 2  Cluster Generation System (Illumina, San Diego, CA) and HiSeq 3000/4000 SE Cluster Kit were used to generate hundreds of millions of single-molecule DNA copies of the original libraries generated during the library preparation steps. Following successful cluster generation, the patterned flow cell containing clusters in the nanowells was transferred to an Illumina HiSeq3000 Sequencing System (Illumina, San Diego, CA) for 100 nucleotide paired-end sequencing-by-synthesis (SBS) using a HiSeq3000/4000 SBS kit.