Computational protocol: Flexible selection of diversified Na+/K+-ATPase α-subunit isoforms for osmoregulation in teleosts

Similar protocols

Protocol publication

[…] The total RNA of medaka and eel were extracted using Isogen (Wako Pure Chemical Industries, Osaka, Japan), reverse transcribed to cDNA libraries using TruSeq RNA Sample Preparation v2 (Illumina Inc, CA), and sequenced by Illumina HiSeq 2500 (101 bp, paired-end) in the Laboratory of Functional Genomics, The University of Tokyo, according to the manufacturer’s protocols. RNA-seq of the gill and intestine samples from medaka was performed as outlined previously []. For eel RNA-seq, the sequenced reads were mapped to the Japanese eel genome [] using TopHat (version 2.0.9) []. The mapped reads were pooled for each condition, and genome-guided transcriptome assembly was performed to reconstruct the eel transcripts using Cuffilink version 2.1.1. The assembled transcripts were merged using Cuffmerge, and the merged transcripts were used for quantifying gene expression levels.For each eel transcript, open reading frames (ORFs) were predicted using EMBOSS getorf (version 6.6.0) [] with the parameter “-minsize 300”. Then, for each gene, the longest ORF among ORFs predicted from all transcripts belonging to the gene was selected, and the translated amino acid sequence of the ORF was used for the following blast search. The reciprocal blast search was performed using amino acid sequences of medaka and eel using BLASTP in NCBI-BLAST+ (version 2.2.29+) [] with the parameter “-evalue 1e-5”. Longest amino acid sequence for each medaka gene in Ensembl annotation (release 74) was used. Reciprocal BLAST best hits (RBBH) in terms of E-value were defined as RBBHs between medaka and eel. Gene annotation of eel was guided using the medaka genome as reference database with RBBH. Only genes with at least 10 reads in at least two samples were used in the following analysis, and low-count genes were removed. The relative gene expression was normalized using the iDEGES method implemented in the TCC package (version 1.2.0) []. Transcriptome of intestine and gill in medaka and eel were deposited in DDBJ database with accession number DRA004257 and DRA004258 respectively. [...] Initially, we searched for putative NKA sequences in the draft genome of Japanese eel using BLASTn, and obtained independent scaffolds (scaffold 12167, 20013, 2250, 15826, 2700, and 8515) and transcript data (t20531, t13768, t17728, and t10968) []. Total RNA was extracted from the frozen tissues using Isogen, treated with DNase I (Life Technologies, Grand Island, NY, USA) to remove genomic DNA, and subsequently reverse transcribed into cDNA by iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc, Hercules, CA, USA) according to the manufacturer’s protocols. To obtain full-length cDNA sequences, specific primers were designed on various regions of the predicted sequences and 3′-RACE was performed to obtain the unknown sequences and 3′-untranslated regions (UTRs). 3′-RACE cDNA templates were prepared from FW and SW eel gills using the SMART cDNA Cloning kit. Long distance PCR was performed to amplify the 3′-ends of NKA isoforms using a KOD plus reagent kit (Toyobo, Osaka Japan) according to the high GC reaction profiles of the manufacturer’s protocol. All sequencing reactions were performed using BigDye Terminator 3.1 cycle sequencing kit (ThermoFisher Scientific, Waltham, MA, USA) according to the company protocols.The deduced protein sequences of representative vertebrate NKA α-subunits were collected from cloning and BLAST search from Ensembl release 84 [] and NCBI nucleotide databases [] and were used to reconstruct the phylogenetic relationship. The sequences were aligned using MUSCLE with default settings in MEGA version 6, and the best protein model was searched and subsequently used in the phylogenetic analysis. Phylogenetic trees were constructed using the maximum likelihood method in MEGA version 6 based on the LG model []. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.3816)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 3.5494 % sites). Bootstrap tests were performed with 1000 replicates to verify the robustness of the phylogenetic relationships.Synteny analysis was performed among the neighbor orthologous genes of atp1a1 (α1), atp1a2 (α2), and atp1a3 (α3) among Japanese eel, medaka, tilapia, and zebrafish to reveal the genomic organization of different isoforms generated by independent and/or genome duplications. […]

Pipeline specifications

Software tools TopHat, Cufflinks, EMBOSS, BLASTP, BLASTN, MUSCLE, MEGA
Databases Full Length cDNA
Applications Phylogenetics, RNA-seq analysis, Nucleotide sequence alignment
Organisms Oryzias latipes
Chemicals Potassium, Sodium