Iso-Seq analysis of Miscanthus sinensis plants
Iso-Seq analysis of Miscanthus sinensis plants
RNA was extracted from 2 cm middle fully expanded leaf segments. Tworeplicates per genotype were collected. Material was frozen in liquid nitrogen and stored at −80 °C. Frozen samples were homogenized using TissueLyser II (Qiagen, Hilden, Germany).  Plants were first grown under greenhouse conditions (25°C day / 19°C night) and then subjected to either a constant control environment or a chilling treatment. Maternal plants were grown from 5 cm rhizome segments, each planted in 40 L containers filled with a 1:1 peat:sand mixture. A standardized nutrient solution (100 mg/kg N, 50 mg/kg P, 200 mg/kg K, 2 mg/kg Fe, 10 mg/kg Zn, 10 mg/kg Ca) with daily watering was implemented. Collected plantlets were initially cultivated in the greenhouse conditions under a 12-hour photoperiod using white fluorescent lighting with photosynthetically active radiation (PAR) at 750 μmol m⁻² s⁻¹. Day/night temperatures were held at 25/19°C, with 60% ± 10% of relative humidity. At the five-leaf stage, 20 individuals per genotype were relocated to a cultivation chamber at the Wielkopolska Centre for Advanced Technologies (Poznań, Poland). Light intensity, photoperiod and temperature were matched to the greenhouse setting to facilitate acclimation.  Frozen samples were homogenized using TissueLyser II (Qiagen, Hilden, Germany). RNA was extracted using an automated method, Maxwell® RSC Plant RNA Kit. The concentration and purity of RNA was measured using the Nanodrop 1000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). RNA integrity was determined using Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Only RNA samples with 260/280 ratio higher than 1.8, 260/230 ratio higher than 2.0, and RIN (RNA integrity number) values higher than 8.0 were used for the RNA-seq analyses.  Iso-Seq library preparation was done using PacBio full-length cDNA library preparation kit according to the manufacture's protocol (Pacific Biosciences of California, Inc., Menlo Park, CA, USA) by the sequencing service provider. 