Combined lrRNAseq from Daphniphyllum macropodum
Tissue was harvested onto dry ice. DNA-depleted RNA was isolated using a Direct-Zol RNA miniprep kit (Zymo Research). RNA quantity and quality were assessed using the Agilent Tapestation system with RNA nano screentape. For full length cDNA sequencing, libraries were prepared (and sequenced by the Bioscience Technology Facility at the University of York) from 100 ng total RNA per species using the Oxford Nanopore Technologies (ONT) PCR cDNA barcoding library prep kit SQK-PCB109, including a 12 cycle PCR amplification of full length cDNA, with unique barcodes for each sample. Following cDNA amplification, equal quantities of cDNA from each species were pooled into final 100 fmol cDNA pool for adapter ligation and loading onto Oxford Nanopore R9.4.1 flow cells and sequencing on an ONT promethION instrument running MinKNOW version 20.06.9. Basecalling and barcode demultiplexing were performed using ONT's guppy basecaller software version 4.0.11 and quality-checked with PycoQC.