RNA-Seq of Salt Stress Applied Oryza sativa shoot
Approximately 5 microgram of total RNA was used to purify mRNA using the Ribo-Zero Gold rRNA Removal Kit (Illumina, San Diego, USA, cat. MRZG12324). The purified RNA was fragmented into smaller pieces using the NEBNext Magnesium RNA Fragmentation Module (NEB, USA, cat. E6150S), which employs divalent cations under high-temperature conditions. The fragmented RNA was then reverse-transcribed into cDNA using SuperScript II Reverse Transcriptase (Invitrogen). Second-strand cDNA synthesis was performed with E. coli DNA polymerase I (NEB, cat. M0209, USA), RNase H (NEB, cat. M0297, USA), and dUTP Solution (Thermo Fisher, cat. R0133, USA), producing double-stranded cDNA labeled with U. The cDNA was subsequently purified, followed by end repair, dA tailing, adaptor ligation, and DNA fragment enrichment. The final cDNA library, with an average insert size of approximately 300 bp, was subjected to paired-end sequencing (2*150 bp) on the Illumina Novaseq 6000 platform, adhering to the manufacturer's protocol.