RNA isolation, Library preparation, and Illumina Novaseq 6000 Sequencing
Transcriptome sequencing was performed by Meiji biotechnology co., LTD (SRA: SRP253018). Total RNA was extracted from the discus fish skin and brain tissue using TRIzol® Reagent according the manufacturer’s instructions (Invitrogen, Carlsbard, CA, USA) and genomic DNA was removed using DNase I (TaKara). Then the integrity and purity of the total RNA quality was determined by 2100 Bioanalyser (Agilent Technologies, Inc., Santa Clara CA, USA) and quantified using the ND-2000 (NanoDrop Thermo Scientific, Wilmington, DE, USA). Only high-quality RNA sample (OD260/280= 1.8~2.2, OD260/230≥2.0, RIN≥8.0, 28S:18S≥1.0, >2μg) was used to construct sequencing library. RNA purification, reverse transcription, library construction and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) according to the manufacturer’s instructions (Illumina, San Diego, CA).The discus fish skin and brain RNA-seq transcriptome libraries were prepared using Illumina TruSeqTM RNA sample preparation Kit (San Diego, CA). Total RNA was separated by oligo-dT-attached magnetic beads and then fragmented by fragmentation buffer. Taking these short fragments as templates, double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA) with random hexamer primers (Illumina). Then the synthesized cDNA was subjected to end-repair, phosphorylation and ‘A’ base addition according to Illumina’s library construction protocol. Libraries were size selected for cDNA target fragments of 200–300 bp on 2% Low Range Ultra Agarose followed by PCR amplified using Phusion DNA polymerase (New England Biolabs, Boston, MA) for 15 PCR cycles. After quantified by TBS380, two RNAseq libraries were sequenced in single lane on an Illumina Novaseq 6000 sequencer (Illumina, San Diego, CA) for 2×150bp paired-end reads.