title: RNA-Seq of yellow catfish: spleen of the normal fish
design: Total RNA was extracted from the spleen tissue using Trizol reagent (Invitrogen) following the manufacturerĄŻs instructions, with an optional on-column DNase digestion step included. RNA quantity, integrity and purity were measured by gel electrophoresis and spectrophotometry (Nano-Drop ND-1000, Thermo Scientific, USA). Equal amounts of RNA from five fish belonging to the same group were pooled, obtaining two RNA samples for cDNA synthesis. Two RNA-Seq libraries were constructed with the Illumina TruSeq RNA Sample Preparation Kit (Illumina) according to the procedures. Briefly, polyA+ RNA was purified from total RNA using selection on oligo-dT-attached magnetic beads and fragmented to an average size of 200 bp using the fragmentation reagent. Thereafter, the first-strand cDNA was synthesized using random hexamer primers and reverse transcriptase, followed by the second-strand cDNA synthesis using DNA Polymerase I and RNase H. After end reparation and 3ĄŻ-end poly(A) addition, double-stranded cDNA was ligated to sequencing adaptors and subsequently purified. Finally, the suitable cDNA fragments were enriched by PCR amplication to create the final cDNA library, which was sequenced via 2ĄÁ100 paired-end sequencing on Illumina HiSeq 2000 platform (Sangon Biotech Co., Ltd, Shanghai, China).