2.2. RNA extraction, library preparation and sequencing
Total RNA was extracted using RNA Plant Mini Spin (Qiagen) kit according to the manufacturer protocol. Adequate RNA quality and quantity of RNA samples were ensured by Tapestation (Agilent) analysis using High Sensitivity RNA screening tape kit and Qubit 4 fluorimeter using HS RNA Assay kit. The purified total RNA was used for sequencing library preparation. Long-read native RNA libraries were prepared from 50 ng of poly(A)-tailed mRNA per sample using Direct RNA Sequencing Kit SQK-RNA002 (Oxford Nanopore Technologies) according to the manufacturer’s protocol. To remove rRNA from total RNA, NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England Biolabs) was used. In the first step of library preparation SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) was used to synthesize the strand complementary to RNA and thus to prepare RNA-cDNA hybrid. In the next step sequencing adapters were attached using T4 DNA Ligase 2M U/ml (New England Biolabs) together with NEBNext® Quick Ligation Reaction Buffer. The libraries were quantified with Qubit dsDNA HS Assay Kit (ThermoFisher) and sequenced using MinION MK1C portable device (ONT) and FLO-MIN 106 Flow Cells R.9.4.1 (ONT) prepared for sequencing with Flow Cell Priming Kit EXP-FLP002 (ONT). The raw reads were basecalled using Dorado 0.4.3 (ONT) using the rna002_70bps_hac@v3 model on the NVIDIA RTX4090 GPU. The raw reads were deposited in the ENA EMBL-EBI database at the following numbers PRJEB72691.
Total RNA for short read procedure was extracted using RNA Plant Mini Spin (Qiagen) kit according to the manufacturer protocol. Adequate RNA quality and quantity of RNA samples were ensured by Tapestation (Agilent) analysis and High Sensitivity kit. The purified total RNA was used for sequencing library preparation. Short-read RNA-seq libraries were prepared using Truseq RNA library with Ribo-Zero option and sequenced using Illumina NovaSeq 6000 platform at Macrogen Inc. (Seoul, Korea). The raw reads from were deposited in the ENA EMBL-EBI database at the following numbers PRJEB72692.