Computational protocol: Transcriptome Analysis and Postprandial Expression of Amino Acid Transporter Genes in the Fast Muscles and Gut of Chinese Perch (Siniperca chuatsi)

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Protocol publication

[…] Total RNA was extracted from the fast muscles of adult Chinese perch by using the RNA extraction KitII(Takara Biotechnology, Dalian, China) and beads with oligo (dT) were used to isolate poly(A) mRNA. Fragmentation buffer was added to disrupt the mRNA to produce short fragments and they were then used as templates. A random hexamer primer was used to synthesize the first-strand cDNA. Second-strand cDNA was synthesized using a buffer containing dNTPs, RNaseH, and DNA polymerase I. The short fragments were then purified using the QiaQuick PCR extraction kit(Takara Biotechnology, Dalian, China) and resolved with an elution buffer (EB buffer) for end reparation and by adding poly(A). Further, the short fragments were connected with sequencing adapters. After agarose gel electrophoresis, suitable fragments were selected as templates for polymerase chain reaction (PCR) amplification. Finally, the library was sequenced using Illumina HiSeq™ 2000.Sequencing was conducted at the Beijing Genomics Institute and transcriptome de novo assembly was performed using the short reads assembly program Trinity []. Trinity was first combined to reads with a certain length of overlap to form longer fragments or contigs. The reads were then mapped back to the contigs. Paired-end reads can detect contigs from the same transcript as well as the distances between these contigs[]. Finally, Trinity was connected to the contigs, and sequences that could not be extended on either end were prepared. Such sequences were defined as unigenes. Unigene sequences were first aligned by using BLASTX to search protein databases, such as NR, SwissProt, Kyoto Encyclopedia of Genes and Genomes (KEGG), and COG (e < 0.00001), and then aligned by using BLASTN to search the nucleotide database NT (e < 0.00001). […]

Pipeline specifications

Software tools Trinity, BLASTX, BLASTN
Databases KEGG
Application RNA-seq analysis
Organisms Siniperca chuatsi
Chemicals Amino Acids