maize leaves with VB1
In this project, Oligo(dT) magnetic beads were used to enrich the polyA-tailed mRNA from total RNA. Ion fragmentation was employed to break the RNA into fragments of approximately 300bp in length. The choice of 300bp fragments was based on the fixed length of the adapters used in the subsequent steps. If the fragmented RNA is too short, it would result in a higher proportion of adapter sequences, thereby reducing the proportion of valid data. Conversely, if the fragmented RNA is too long, it would hinder cluster generation during sequencing. The first-strand cDNA was synthesized using a 6-base random primer and reverse transcriptase, with the RNA as the template. The second-strand cDNA was then synthesized using the first-strand cDNA as the template. After library construction, PCR amplification was performed to enrich the library fragments, followed by library selection based on fragment size, targeting a library size of 450bp. The library was then subjected to quality control using the Agilent 2100 Bioanalyzer, including evaluation of the total and effective concentration. Based on the effective concentration and the required amount of data for the library, libraries with different index sequences (each sample with a unique index) were mixed proportionally. The mixed libraries were diluted to a final concentration of 8nM and converted into single-stranded libraries through alkaline denaturation. Following RNA extraction, purification, and library construction, the libraries were subjected to paired-end (PE) sequencing using next-generation sequencing (NGS) technology on the Illumina sequencing platform.