Illumina NovaSeq 6000 paired end sequencing; RNA-seq of part of the root system of barley genotypes grown in an agricultural soil under controlled conditions
RNA-seq of part of the root system of barley genotypes grown in an agricultural soil under controlled conditions
At early stem elongation plants were uprooted from their pots and roots were washed from the adhering rhizosphere soil using vortexing and mild saline buffer prior nucleic acid extraction. Plants were grown in a randomized design in a glasshouse at 18/14 °C (day/night) temperature regime with 16 h daylight that was supplemented with artificial lighting to maintain a minimum light intensity of 200 µmol quanta m−2 s−1. RNA was extracted from individual root systems with the Macherey-Nagel™ NucleoSpin™ RNA, Mini Kit (Thermo Fisher, USA) following the manufacturer's protocol, including the Plant RNA Isolation Aid Invitrogen (Thermo Fisher, USA) for the sample lysis step using 90 µL of Plant RNA Isolation Aid, mixed with 870 µL of RA1 buffer and 40 µL of dithiothreitol (DTT). RNA quality was assessed using an Agilent 2100 Bioanalyzer or TapeStation (Agilent, USA). Samples for sequencing were selected based on microbiota profiles and quality of the RNA sample Total RNA (300 ng) was further purified using the NEBNext mRNA Magnetic Isolation Module (NEB). Library preparation was carried out using the NEBNext® Ultra™ II Directional RNA Library Prep with Sample Purification Beads and indexed with NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs set 1).