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.