RNA-seq of Narcissus cv. Tete a Tete, Pooled
RNA-seq of Narcissus cv. Tete a Tete, Pooled BO-16, F-2, LB-2, LT-6, SB-1.Preparation and sequencing of the PacBio SMRT sequencing library was performed by QB3 Genomics, UC Berkeley, Berkeley, CA, RRID:SCR_022170 on the PacBio Sequel II System instrument using Sequel II 2.1 binding kit with a movie time of 30 hours. RNA concentration was normalized to < 5 ng/L using the Qubit HS RNA Kit, and then capillary electrophoresis was performed using the Agilent Bioanalyzer 2100 instrument, using the Eukaryotic Total RNA Pico Assay, following manufacturers instructions, with a  2 min denaturation at 70C followed by a 2 min cold-shock on ice in a freezer. Preparation of the PacBio SMRT sequencing library was performed as described in the Iso-SeqTM Template Preparation for Sequel Systems manual using the NEBNext Single Cell/Low Input cDNA Synthesis & Amplification and Pacific Biosciences Iso-Seq Express Oligo Kit with barcoded primers listed in the manufacturer supplied protocol to perform first-strand cDNA synthesis (standard purification) and PCR amplification. Barcoded cDNA from different samples was pooled in equimolar quantities and a single library was prepared from this pooled cDNA using the SMRTbell Express Template Prep Kit 2.0 following the standard protocol. Raw data from this experiment were processed using the default parameters for the Iso-Seq 2 pipeline available via SMRT link software (PacBio, https://github.com/PacificBiosciences/IsoSeq/blob/master/isoseq-clustering.md). This processing generated a final sequence file of 432,086 high quality transcript isoforms. We then used CD-HIT-EST (2) with a cut-off at 97% identity to remove potentially redundant transcript sequences, which resulted in a final total of 90,359 unique transcripts. The quality of the assembly was assessed by mapping short read Illumina sequencing reads (see Methods) to the long read transcriptome assembly (91.2% overall alignment rate) and BUSCO (89.1% completeness).