GSM8164065: embryo10DAA; treated; replicate 1; Oryza sativa; OTHER
Total RNA isolation: total RNA was isolated according to TRIzol reagent manufacturer's protocol, followed by isopropanol precipitation. PolyA+ RNA isolation: Dynabeads™ mRNA Purification Kit (Invitrogen™) was used for polyA+ RNA enrichment, from the purified total RNA. With 10 ng synthetic RNA oligos carrying 0%, 20%, 40%, 60%, 80%, or 100% pseudouridine, 5′-phosphorylation were conducted with T4 polynucleotide kinase (PNK) (catalog no. EK0032, Thermo Fisher Scientific). To perform 3′-adapter ligation, 10 µl 5′-phosphorylated RNA fragments were incubated with 1.0 µl 20 µM RNA 3′ SR Adapter (5′App-NNNNN AGATCGGAAGAGCACACGTCT-3SpC3, with ATCACG as the inline barcode) at 70 °C for 2 mins and placed immediately on ice. Then, 2.5 µl 10× T4 RNA Ligase Reaction Buffer (catalog no. M0373L, NEB), 7.5 µl PEG8000 (50%), 1 µl SUPERase•In RNase Inhibitor and 2 µl T4 RNA Ligase 2 truncated KQ (catalog no. M0373L, NEB) were added to the RNA–adapter mixture48,49. The reaction was incubated at 25 °C for 2 h followed by 16 °C for 10 h. The reaction was further diluted to 47 µl with nuclease-free water, and the excessive adapters were removed with 2 µl 5′