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Sample GSM2409495

Query DataSets for GSM2409495

Status

Public on Jan 01, 2018

Title

T2_0_K_mRNA

Sample type

SRA

Source name

control sample, were injected with PBS at the same dosage as treated sample.

Organism

Carassius gibelio

Characteristics

tissue: kidney
development: one year old

Treatment protocol

After seven days of acclimation, fish were inoculated by intraperitoneal injection with Cyhv-2 suspended in PBSat a dose of 1 ×106 TCID50/g which was applied and verified by previous challenge experiments5. As control, fish were injected with PBS at the same dosage. After the injection, all the fish were fostered under the same and suitable condition, fed with a diet according to standard feeding scheme, and continuously observed for collecting moribund animals.

Growth protocol

Healthy silver crucian carp for the present experiment (approximately 10 cm in body length) were obtained from the SHOU experimental fish breeding farm. Initially, fish were temporarily reared in at 23 °C for adaptation.

Extracted molecule

total RNA

Extraction protocol

RNA preparation, library construction and high-throughput sequencing were performed by LC Sciences (Houston, TX, USA). The experimental procedure is introduced as follows: total RNAs were extracted using Trizol reagent (Invitrogen, CA, USA) following the instruction of the product manual. The total RNA quantity and purity were analyzed by using Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number higher than 7.0. The total RNA with lowest quality was not used for further study from the pre-receptive endometrium samples and receptive endometrium samples, respectively. For sRNA-seq experiment, approximately 1 μ g of total RNA was subjected to construct sRNA library according to the protocol of TruSeq™ Small RNA Sample Prep Kits (Illumina, San Diego, USA). Then, single-end sequencing (50 bp) was performed on the Illumina Hiseq2500 platform following the vendor’s recommended protocol. For RNA-seq experiment, approximately 10 μ g of total RNA was used to enrichment of poly (A)-tailed mRNAs with poly-Toligo attached magnetic beads (Invitrogen, MA, USA). Following purification, the mRNAs were fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments were reverse-transcribed to produce the final cDNA library according to the protocol of the mRNA-seq sample preparation kit (Illumina, San Diego, USA). Each PE and RE library was constructed by pooling 9 homogenized total RNAs from the endometrium samples. Then, paired-end sequencing of the PE and RE libraries was performed on the Illumina Hiseq2500 (LC Sciences, USA) following the vendor’s recommended protocol. The length of the reads was 100 bp, and the average insert size for the paired-end libraries was 179 bp (the length of the adapter was 121 bp). Pre-treatment of the sRNA-seq. First, the sequencing adapters were removed and the short reads containing ambiguous base “N” were discarded. Then, in order to allow cross-library comparison, read count normalization was performed. Specifically, the normalized read count (in RPM, reads per million) of a short sequence from a sequencing library was calculated by dividing the raw count of this sequence by the total counts of the library, and then multiplied by 10^6.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

For the mRNA analyze, the raw paired end (PE) reads were cleaned by removing adapter sequences, empty reads, and low quality sequences (reads with over 10% unknown base pairs ‘N’). The reads obtained were randomly decomposed into overlapping k-mers (default k = 25) for assembly by using Trinity software69. To identify transcripts homologous to those in the other species, a locally installed BLAST all program70 was utilized to search the assembled transcripts against the sequences in NCBI NR protein database (http://www.ncbi.nlm.nih.gov/protein/)71 and the Swissprot database (http://www.uniprot.org/)72 with E-values lower than 1e-10. Genes were tentatively annotated according to the best hits against known sequences. COG73 and KEGG74 annotation systems were employed to analyze the biological pathways that the assembled transcripts were involved in. For expression level calculation, Bowtie (version 0.12.7)75 was utilized to map RNA-seq reads to all the assembled transcripts by using the “single-end” method and the parameter “-v 3 -a – phred 64-quals” (allowing one read to be mapped to multiple transcripts). The perfectly mapped reads counts were retained for expression level calculation by using the following formula: Expression level of a transcript (RPKM; reads per kilobase of exon model per million mapped reads) = Number of the reads mapped to the transcript / [Total number of the reads mapped to all the transcripts (in million) × the length of the transcript (in kilobases)]
Genome_build: none de novo sequencing
Supplementary_files_format_and_content: 1_Expressed_miRNA.xlsx, 2_gene_expression.xlsx,3_Trinity_gene.fasta, format and content :fasta,xlsx, assembly sequences and expression data.

Submission date

Nov 29, 2016

Last update date

May 15, 2019

Contact name

jianfei lu

E-mail(s)

[email protected]

Organization name

Shanghai Ocean University

Department

Freshwater Fishery Germplasm Resources