RNA Sequencing Library Preparation Protocol

An RNA sequencing library was prepared using the TruSeq™ RNA Sample Preparation Kit from Illumina (San Diego, CA) following the manufacturer’s recommendations. In brief, mRNA was purified via Poly (A) selection with oligo(dT) cellulose. and then fragmented in fragmentation buffer. Continually, double-stranded cDNA was synthesized using a Super Script Double-Stranded cDNA Synthesis Kit (Invitrogen, CA) with random hexamer primers (Illumina). The synthesized cDNA was subjected to end-repaired, phosphorylation and the A-tailed according to library-construction protocol of Illumina. Libraries were size-selected for cDNA target fragments of 300 base pairs (bp) on a 2% low-range ultra agarose gel, followed by PCR amplification. Finally, the amplified fragments were sequenced with an Illumina HiSeq Xten/NovaSeq 6000 sequencer according to the manufacturer’s instructions^{21 (link)}. The sequencing data have been deposited in the NCBI Sequence Read Archive (SRA), and are accessible through the accession number PRJNA727442.