RNA-seq of Hordeum vulgare (cultivar Nure)
Approximately 30 mg of plant tissues were placed in a 1.5 ml Eppendorf LoBind tube containing glass beads (1.7-2.1 mm diameter, Carl Roth, Karlsruhe, Germany) and 100 l of TRI-Reagent (Zymo Research). The Eppendorf tube was firmly attached to a SILAMAT S5 vibrator (Ivoclar Vivadent, Schaan, Liechtenstein) in order to disrupt and homogenize the tissue for 2x15s. Total RNA was extracted using Direct-zol RNA MiniPrep System (Zymo Research) according to the manufacturers protocol. The RNA Integrity Numbers and RNA concentration were determined by RNA ScreenTape system with 2200 Tapestation (Agilent Technologies, Santa Clara, CA, USA) and RNA HS Assay Kit with Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively. For Gene Expression Profiling (GEx) library construction, QuantSeq 3 mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen GmbH, Wien, Austria) was applied according to the manufacturers protocol. The quality and quantity of the library was determined by using High Sensitivity DNA1000 ScreenTape system with 2200 Tapestation (Agilent Technologies, Santa Clara, CA, USA) and dsDNA HS Assay Kit with Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively. Pooled libraries were diluted to 1.8 pM for 1x86 bp single-end sequencing with 75-cycle High Output v2 Kit on the NextSeq 550 Sequencing System (Illumina, San Diego, CA, USA) according to the manufacturers protocol. By using longer reads QuantSeq FWD allows to exactly pinpoint the 3 end of poly(A) RNA and therefore obtain accurate information about the 3 UTR. Using this sequencing fragments of coding sequences are 260-300 bp long in average.