title: Illumina NovaSeq 6000 paired end sequencing; High-throughput RNA sequencing in crown tissue of barley sdw1 near-isogenic lines
design: High-throughput RNA sequencing in crown tissue of barley sdw1 near-isogenic lines
library_construction_protocol: Barley crown tissue was sampled for gene expression analysis using mRNA-seq in three biological replications at two time points: 1 d, tillering stage (23-26 of BBCH code), and 10 d, ten days after the first day. Each replication consisted of crown samples collected from three plants per pot.  Plants were grown in pots (H-LSR 4.5 L; 21 cm in diameter and 20 cm in height), with eight seeds per pot, filled with a mixture of loamy soil and peat (3:1, w/w) under controlled conditions (60% air humidity, 234 µmol m−2 s−1 PAR irradiance; Apollo 8 LED Grow Light). Two temperature regimes were applied: (i) optimal (control) temperature (OT) of 8/16 °C (night/day) from sowing to the end of tillering and then 12/20 °C to maturity; (ii) elevated temperature (HT) of 28 °C from sowing to the end of tillering and then 12/20 °C as in the control. An 8/16 h (dark/light) photoperiod was maintained, while soil moisture was maintained above 70% field water capacity, controlled by the daily weighing of each pot. Total RNA was extracted using TRI Reagent® RT (Molecular Research Center, Inc., Cincinnati, OH, USA) according to the manufacturer's protocol and treated with DNase I during RNA purification. The quality and quantity of RNA were verified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) using the following criteria: 2.0 for 260/280 and 260/230 ratios. RNA integrity number (RIN) of samples sufficient for sequencing (≥ 8) was confirmed using an Experion™ electrophoresis station (Bio-Rad Laboratories, Hercules, CA, USA).  cDNA library construction (TruSeq stranded mRNA) was done by Macrogen (Seoul)