Banana Fruit RNA
A total of 3 g of RNA per sample was utilized as input material for the RNA sample preparations. Initially, ribosomal RNA was removed using the Epicentre Ribo-zero rRNA Removal Kit (Epicentre, USA), and the rRNA-free residue was subsequently cleaned up through ethanol precipitation. Following this, sequencing libraries were generated from the rRNA-depleted RNA using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, USA), adhering to the manufacturers recommendations. To select cDNA fragments preferentially ranging from 150 to 200 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). Next, 3 l of USER Enzyme (NEB, USA) was applied to the size-selected, adaptor-ligated cDNA at 37 C for 15 minutes, followed by a 5-minute incubation at 95 C prior to PCR. PCR amplification was conducted using Phusion High-Fidelity DNA polymerase along with Universal PCR primers and an Index (X) Primer. Finally, the products were purified using the AMPure XP system, and library quality was assessed using the Agilent Bioanalyzer 2100 system. To ensure the quality of the library, a qPCR method was employed to accurately quantify the effective concentration of the library, which was determined to be 2 nM. The clustering of the index-coded samples was performed on a cBot Cluster Generation System utilizing the TruSeq PE Cluster Kit v3-cBot-HS (Illumina) in accordance with the manufacturers instructions. Following cluster generation, the libraries were sequenced on an Illumina HiSeq 2500 platform, resulting in the production of 100 bp paired-end reads.