Barley parent biological replicate 3
Total RNA from 3 plants per genotype were isolated using innuPREP Plant RNA Kit (Innuscreen GmbH, Berlin, Germany). Total RNA concentration were measured using Qubit 4 fluorometer (Thermo Fisher Scientific, USA) with Equalbit RNA BR Assay Kit (Vazyme, Nanjing, China) and RIN value was measured using Bioanalyzer 2100 System (Agilent, Santa Clara, CA, USA) with RNA 6000 Nano Kit (Agilent, Santa Clara, CA, USA). rRNA was removed from the total RNA samples using Ribo-off rRNA Depletion Kit Mega (Plant) (Vazyme, Nanjing, China), following the manufacturers instructions. Following rRNA removal, the remaining RNA was used to prepare RNA-seq libraries for Illumina sequencing using the VAHTS Universal V10 RNA-seq Library Prep Kit (Premixed Version) (Vazyme, Nanjing, China), following the manufacturers instructions. RNA-seq library clean-up and size selection were subsequently performed using NucleoMag NGS Clean-up and Size Select magnetic beads (Macherey-Nagel, Germany), resulting in a final library size of 400-500 bp, corresponding to an insert size of 280-380 bp. The purified size selected libraries were subsequently amplified using the VAHTS Maxi Unique Dual Index Primers Set 1 (Vazyme, Nanjing, China) following the manufacturers protocol. The resulting indexed libraries were quantified using the Qubit 4 fluorometer (Thermo Fisher Scientific, USA) with Equalbit 1 X dsDNA HS Assay Kit (Vazyme, Nanjing, China), and quality controlled using the Bioanalyzer 2100 System (Agilent, Santa Clara, CA, USA) with High Sensitivity DNA Kit (Agilent, Santa Clara, CA, USA). Libraries were then pooled in equimolar amounts and, to ensure optimal cluster generation on the Illumina platform, the final pool was spiked with 10% PhiX control DNA (Illumina, San Diego, CA, USA). The pooled libraries were subsequently sequenced on a NextSeq 2000 (Illumina, San Diego, CA, USA) with a P1 300-cycle flow cell kit (Illumina, San Diego, CA, USA) to generate 150 bp paired-end reads.