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Sample GSM364844

Query DataSets for GSM364844

Status

Public on Nov 24, 2009

Title

Muscle_animal_4365

Sample type

SRA

Source name

Longissimus dorsi muscle

Organism

Sus scrofa

Characteristics

longissimus dorsi muscle, animal: 4365, gender: male, age: 2 years

Extracted molecule

total RNA

Extraction protocol

Total RNA was purified using the mirVanaTM miRNA isolation kit (Ambion) following the manufactures protocol and 10 µg total RNA from each sample was used for library constructions following the protocol supplied with the Small RNA Sample Prep Kit (Illumina, protocol version 1004239_RevA, March 2008) with minor modifications. Briefly, the 18-30 base pair fraction of total RNA was excised from 15 % Criterion Tris-Borate-EDTA (TBE)-Urea gels (Bio-Rad) stained with SYBR Safe DNA gel stain (Invitrogen) and visualised using a Dark Reader Transilluminator (Clare Chemical Research). Samples were electrophorised on individual gels throughout the library preparation to avoid cross-contamination. The small RNA was purified, ligated with the SRA 5´ and 3´ adaptors, reverse transcribed and amplified as recommended by the manufacturer. The 90 base pairs PCR products were purified on 4 % low range agarose gels (manufacturer) using the QIAquick Gel Extraction Kit (QIAGEN), eluted in 30 µL elution buffer and dried to a volume of 10 – 15 µL using a SC210A SpeedVac® Plus (ThermoSavant). The concentrations of the libraries were determined using a NanoDrop ND-1000 Spectrophotometer (manufacturer) and the size and purity were determined using an Agilent 2100 Bioanalyzer in combination with the Agilent DNA 1000 Kit. The seven libraries were diluted in buffer EB (QIAGEN) to 10 nM, denaturated with 2 N NaOH to a final DNA concentration of 0.5 nM, diluted to 2 pM with pre-chilled Hybridization buffer (Illumina) and loaded in individual lanes into a 1.0 mm flowcell together with a single lane of a 2 pM PhiX control library (Illumina). Clustering and 36 cycle sequencing were conducted on an Illumina Genome Analyzer (version I) using the Small RNA sequencing primer in combination with clustering and sequencing kits supplied by Illumina. The resulting images were transferred to the pipeline computer (DELL PowerEdge 2900, 8 cores (2 x 4 CPU, 2.6 GHz, 16 GB RAM, 4.7 TB hard drive) during the run using robocopy and analysed using the Genome Analyzer Pipeline Software (version 1.0, Illumina) generating the raw fastq files.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina Genome Analyzer

Description

Muscle_animal_4365

Data processing

The raw sequences were trimmed to 30 nucleotides, and it was set as a requirement that any sequence must appear at least three times and be present in at least two of the seven libraries. All identical reads within a library were grouped and converted into unique sequences. Reads containing Ns or long tracks (¡Ý8) of As were removed and the sequences were trimmed for adaptor-sequences. To annotate the unique sequences, a Decypher Tera-BLASTN Search was performed against a database of mature miRNAs obtained from miRBase (release 12.0). Hits with a match of 16 or more nucleotides to a miRNA from the database were gathered, and the count of each miRNA was normalized to the total number of sequence reads per lane.

Submission date

Jan 27, 2009

Last update date

May 15, 2019

Contact name

Jakob Hedegaard

E-mail(s)

Jakob.Hedegaard@ki.au.dk

Phone

(+45)89991363

Organization name

Aarhus University, Faculty of Agricultural Sciences