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Status |
Public on Oct 16, 2015 |
Title |
Male Gut Replicate 2 sRNAseq |
Sample type |
SRA |
|
|
Source name |
Male Gut
|
Organism |
Danio rerio |
Characteristics |
gender: Male tissue: Gut genetic background: Wild type - Singapore strain
|
Treatment protocol |
N/A
|
Growth protocol |
Fishes were purchased from a local supplier and acclimatized before tissue extraction.
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA were extracted using mirVana™ miRNA Isolation Kit (AM1560, Life Technologies). Tissues were homogenised in 1.5 ml microfuge tube containing Lysis/Binding buffer provided in the mirVana™ miRNA Isolation using a hand held pestle. Total RNA containing small RNA were purified following the manufacturer protocol. Small RNA libraries were prepared for sequencing using TruSeq Small RNA Sample Preparation Kit ( RS-200-0012, Illumina, Inc.). Libraries were prepared according to manufacturer instructions. Briefly, 1µg of good quality Total RNA per sample was used as starting material. 5’ and 3’ RNA adapters were ligated to each RNA molecule before reverse transcription to create single stranded cDNA. The cDNA was then amplified with PCR using a common primer and a primer containing a unique index sequence. The resulting PCR reactions were electrophoresed on 6% Novex TBE PAGE Gel (Life Technologies) and bands corresponding to adapter-ligated constructs derived from 22-30 nucleotides small RNA fragments were excised from the gel. The small RNA were purified from the excised gel and validated on High Sensitivity DNA chips on a Bioanalyser (Agilent Technologies) before sequencing.
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Library strategy |
miRNA-Seq |
Library source |
transcriptomic |
Library selection |
size fractionation |
Instrument model |
Illumina HiSeq 2000 |
|
|
Data processing |
Illumina Casava 1.8.2 software used for basecalling. The sequenced reads were first subjected to adapter removal through the cutadapt program (Martin 2011). The trimmed reads were then collapsed to remove redundancy and to obtain a unique sequence fasta file through the mapper module of miRDeep2 package (Friedländer et al. 2012). The unique reads fasta file was then put through an elimination pipeline module (Vaz et al. 2010) comprising of a series of sequence similarity searches with the annotated databases. At each step the reads were matched to an annotated database with a maximum of two mismatches. The matched reads were removed and the unmatched ones were further matched to another annotated database, finally culminating into an un-annotated pool of reads that served as a source of novel miRNAs and novel sRNAs. The known miRNA expression profile was generated by using the quantifier module of the miRDeep2 package that gives the read counts for the known miRNAs. The quantifier.pl command line used: perl quantifier.pl -p <zebrafish precursor miRNA fasta file> -m <zebrafish mature miRNA fasta file> -r <unique reads fasta file> -t Zebrafish The raw reads expression profile generated for all the replicates of the samples were subjected to Trimmed Mean of M-values (TMM) normalisation using the Bioconductor package edgeR (Robinson et al. 2010). Genome_build: ZV9 Supplementary_files_format_and_content: 1. 'Danio-rerio-known-miRNA-Rel19-profile-Raw.txt': Tab-delimited text file that includes the raw counts for the known mature miRNA (miRBase Release 19). Supplementary_files_format_and_content: 2. 'Danio-rerio-known-miRNA-Rel19-profile-Normalised.txt': Tab-delimited text file that includes the Trimmed Mean of M-values (TMM) normalised counts for the known mature miRNA (miRBase Release 19). One of the replicate of Heart (MZH008) failed to cluster with the other two replicates on basis of its known miRNA expression profile and hence was not used for further analysis.
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|
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Submission date |
Apr 29, 2014 |
Last update date |
May 15, 2019 |
Contact name |
Vivek Tanavde |
E-mail(s) |
vivek@bii.a-star.edu.sg
|
Organization name |
Bioinformatics Institute, A*STAR, Singapore
|
Department |
Genome and Gene Expression Data Analysis Division
|
Lab |
Expression and Signaling in Mesenchymal and Hematopoietic Stem Cells
|
Street address |
30 Biopolis Street, #07-01, Matrix
|
City |
Singapore |
State/province |
Singapore |
ZIP/Postal code |
138671 |
Country |
Singapore |
|
|
Platform ID |
GPL14875 |
Series (1) |
GSE57169 |
Deep sequencing of small RNA facilitates tissue and sex associated microRNA discovery in zebrafish |
|
Relations |
BioSample |
SAMN02740881 |
SRA |
SRX529128 |