GSM5328913: Valley Oak (SW.786) bud RNA long read sequencing (isoseq); Quercus lobata; RNA-Seq
RNA extractions were performed between November 6-8, 2017 using a modified version of the Conifer RNA prep protocol from the Cronn Lab  (https://openwetware.org/wiki/Conifer_RNA_prep) and the Spectrum Plant Total RNA kit (Sigma, St. Louis, MO, USA).  Plant tissues (100 mg each of leaves, buds and stems) were flash frozen in liquid nitrogen and ground with a mortar and pestle to a fine powder. Powdered tissues were transferred to cold 2mL tubes and 1.8 mL of cold RNA Extraction Buffer + DTT was added. RNA Extraction Buffer consists of 8 M Urea, 3 M LiCl, 1% polyvinylpyrrolidone K-60, 5 mM DTT (add just before use, 1M stock). The tubes were then vortexed for 30 seconds, incubated at 4°C for 30 minutes, then centrifuged at 4°C for 30 minutes at 20,000 rcf. The supernatant was discarded and the pellet was used as the starting material for the Spectrum Plant Total RNA kit, protocol A, adding 750uL of Binding Solution, and performing on-column DNase I digestion. RNA quality and quantity were assessed using the Agilent RNA ScreenTape System on the Agilent 2200 TapeStation system (Agilent Technologies, Santa Clara, CA, USA). RNA from leaves, bud and stems were further prepared following the “Guidelines for Preparing cDNA Libraries for Isoform Sequencing (Iso-SeqTM) User Bulletin” (Pacific Biosciences of California, Inc., 2014) and the “Procedure & Checklist – Iso-Seq™ Template Preparation for Sequel™ Systems” (Pacific Biosciences of California, Inc., 2017).  PolyA+ RNA was extracted from total RNA using the Ambion® Poly(A) Purist™ MAG Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol.  First strand cDNA synthesis was performed using the SMARTer® PCR cDNA Synthesis Kit (Takara Bio, Inc., Kusatsu, Shiga Prefecture, Japan), with three reactions using 3.5uL of PolyA+ RNA per tissue, for a total of nine first strand synthesis reactions.  Each of nine reactions were diluted with 90uL of EB buffer, then pooled according to tissue type.  PCR cycle optimization resulted in the following PCR conditions with 24 x 50uL reactions per tissue type: 95°C for 2 minutes for initial denaturation, then n cycles of 98°C for 20 seconds, 65°C for 15 seconds, 72°C for 4 minutes, where n=10 for leaf cDNA, n=12 for bud cDNA, n=14 for stem cDNA, then 72°C for 5 minutes for the final extension. Twelve reactions per tissue type were pooled for 1X AMPure XP (Beckman Coulter, Inc., Pasadena, CA, USA) bead purification, and 12 reactions per tissue type were pooled for 0.4X AMPure XP bead purification.  Samples were sent to the DNA Technologies & Expression Analysis Core Laboratory at the University of California, Davis for size selection, enrichment, library preparation, and sequencing.  A second bead size selection was performed, 1X and 0.4X (Fraction 1 & 2, respectively) for two size fractions and a size selection of 5-10kb using the BluePippin Size Selection System. Six libraries were made from these different size selections following the “Procedure & Checklist – Iso-Seq™ Template Preparation for Sequel™ Systems” protocol: Library 1:  Leaf full length (Fractions 1 & 2); Library 2:  Bud full length (Fractions 1 &2), Library 3:  Stem full length (Fractions 1 &2); Library 4:  Leaf fraction 3 (5-10kb size selection); Library 5:  Bud fraction 3 (5-10kb size selection); Library 6:  Stem fraction 3 (5-10kb size selection). Bud libraries were pooled for sequencing (5:1, full length:fraction3), leaf libraries were pooled for sequencing (5:1, full length:fraction3), and stem libraries were pooled for seqencing (5:1, full length:fraction3) for a total of three libraries that were each sequenced on a single cell. The cells were loaded on the Sequel by Magbead / v2 SMRT cell / P2.1C2.1 (polymerase and chemistry versions).