RNA-seq of Narcissus cv. Tete a Tete leaf 4, bottom
Daffodil tissues frozen at -80 C were homogenized to a fine powder under liquid nitrogen using a mortar/pestle that were pre-treated with RNAse Zap RNAse Decontamination Solution (Thermo Fisher Scientific). Two separate RNA extraction protocols were used for different daffodil tissues: for root, flower and stem tissue, RNA was extracted from ground tissue using the RNEasy Plant Mini Kit (Qiagen), with DNAse on column digestion. For leaf and bulb scale tissue, RNA was extracted using TRIzol reagemt (Thermo Fisher Scientific) and cleaned up using the RNEasy Plant Mini Kit (Qiagen) with DNAse on column digestion. RNA quantification for each sample was performed via NanoDrop and Qubit HS (high sensitivity) RNA assay kit (Thermo Fisher Scientific), and quality was further assessed by measuring size distribution using an Agilent 2100 Bioanalyzer.. Library preparation for Illumina NovaSeq 6000 S4 PE150 sequencing was performed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs), as per the manufacturer instructions. Individual libraries were barcoded with an internal index set at Novogene. Inserts were size selected with NEBNext beads to have an average insert size of 300-600 bp, and the quality and size distribution of each library was confirmed with analysis using a High Sensitivity DNA chip on a 2100 Bioanalyzer (Agilent). Equimolar amounts of libraries for each sample (28 total) were pooled for paired-end sequencing within a single Novaseq 6000 lane (S4 PE150 sequencing).