Transcriptome ONT cDNA sequencing of Dipsacus fullonum seeds

Achenes (hereafter referred to as seeds) of Dipsacus fullonum were collected at the natural time of dispersion in the years 2021, 2023, and 2024 from 6 different populations. For RNA isolation, seeds were incubated until germination reached 1% of final germination, then frozen in liquid nitrogen and ground into a fine powder. RNA was extracted starting from 50 mg of plant material using the RNeasy PowerPlant Kit with DNase treatment (Qiagen, Hilden, Germany). The quality and integrity of RNA were measured with the Eppendorf BioSpectrometer. For the transcriptome sequencing, we used ONT-Nanopore technologies with 50 ng of total RNA following the cDNA-PCR sequencing protocol (SQK-PCS109, ONT-Nanopore, Oxford, United Kingdom). We considered 4 different treatments: dark, light, alternating temperatures, and alternating temperatures plus light. The samples were loaded on the SpotON flow cells inserted in the MinION Mk18, and then the sequencing was monitored with the MinKnow software for 72 h. The data obtained in the .fast5 format were processed with the software Guppy (4.2.2 version) using a server of Google CoLab in super accuracy (SUP) base calling mode.