title: 22Fe-2
design: For RNA sequencing, a total 1.5 g RNA was used to generate paired-end RNA-seq library. Oligo dT is then utilized to enrich mRNA from total RNA for transcriptome analysis. In summary, mRNA is isolated using the polyA selection method with Oligo dT beads, followed by fragmentation using a fragmentation buffer. Subsequently, double-stranded cDNA is synthesized using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, CA) and random hexamer primers. The synthesized cDNA undergoes end repair, phosphorylation, and adapter ligation. Subsequently, the cDNA library, targeting fragments of 300 bp, is selected on a 2% Low Range Ultra agarose gel and subjected to 15 cycles of PCR amplification using Phusion DNA polymerase (NEB). Following quantification with Qubit 4.0, the sequencing libraries are processed for sequencing on both the NovaSeq X Plus platform (PE150) utilizing the NovaSeq Reagent Kit and the DNBSEQ-T7 platform (PE150) employing the DNBSEQ-T7RS Reagent Kit (FCL PE150) version 3.0.