RNA-Seq of Cynoglossus semilaevis spleen
Total RNA of gills were isolated and purified using TRIzol reagent, quantified with NanoDrop ND-1000, and assessed for integrity with Bioanalyzer 2100. Poly(A) RNA was purified from 1g total RNA using Dynabeads Oligo (dT)25-61005, then fragmented with Magnesium RNA Fragmentation Module. Cleaved RNA fragments were reverse-transcribed with SuperScript II Reverse Transcriptase and synthesized into U-labeled second-stranded DNAs with E. coli DNA polymerase I, RNase H, and dUTP Solution. Adapters were ligated to the ends of each strand, followed by size selection with AMPureXP beads. After treatment with heat-labile UDG enzyme, ligated products were PCR amplified, and the final cDNA library had an average insert size of 30050 bp. Finally, paired-end sequencing (PE150) was performed on an Illumina Novaseq X