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Sample GSM866658

Query DataSets for GSM866658

Status

Public on May 01, 2013

Title

PI3Kalpha TAC 11 LV miRNA Seq

Sample type

SRA

Source name

PI3Kalpha TAC 11 LV RNA

Organism

Mus musculus

Characteristics

genotype: PI3Kalpha mutant
strain: FVB
tissue: heart left ventricle
treatment: TAC

Treatment protocol

LV collected from animals 7 days after sham or TAC operation

Growth protocol

Adult WT or caPI3Kalpha animals

Extracted molecule

total RNA

Extraction protocol

Small RNA libraries were prepared using TrueSeq Small RNA Sample Prep Kits (Illumina) in accordance with the manufacturer’s instructions. Briefly, 3’ and 5’ adapters were sequentially ligated to small RNAs (from 1 µg total RNA), followed by a reverse transcription reaction to create single stranded cDNAs, which were then amplified (by PCR) and barcoded using a common primer and a primer containing unique six-base index sequence. The amplified libraries were size-selected/gel-purified and quantified using Qubit dsDNA HS Assay kit (Life Technologies). Six to eight barcoded libraies were pooled in equimolar (10 nmol/L) amounts and diluted to 8 pmol/L for cluster formation on a single flow cell lane, followed by single-end sequencing on an Illumina HiSeq 2000 sequencer. Messenger RNA (mRNA) libraries were prepared using TrueSeq RNA Sample Prep Kits (Illumina) in accordance with the manufacturer’s recommendations. In brief, 3µg of total LV RNA was twice oligo(dT) selected using poly-T oligo-attached magnetic beads. The poly-A(+) RNA was then eluted, fragmented and reverse transcribed into first strand cDNA using random hexamers, followed by second-strand cDNA synthesis. Double-stranded cDNAs were end-repaired and adenylated (singly) at the 3’ ends. Barcoded adapters containing unique six-base index sequences and T-overhangs were ligated to the cDNA samples prepared from each of the individual mouse LV samples. Individual cDNA libraries were PCR amplified and purified; six barcoded libraries were pooled in equimolar (10 nmol/L) amounts and diluted to 4 pmol/L for cluster formation on a single flow cell lane, followed by single-end sequencing on an Illumina HiSeq 2000 sequencer.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

(for miRNASeq) Sequencing data from samples pooled in the same flow cell lane were separated (demultiplexed) using CASAVA 1.6 software (Illumina). The sequence reads were analyzed using the miRanalyzer program, where the raw sequencing data were transformed and filtered to keep only sequences containing 17-26 bases. Filtered reads were then successively mapped (using Bowtie) to the miRBase22 v.16 mouse database (allowing up to two mismatches), to detect known miRNAs. Read counts are provided.
(for mRNASeq) After demultiplexing the sequencing data, adapter sequences were removed and the individual libraries were converted to the FASTQ format. Sequence reads were mapped to the mouse genome (mm9) with Bowtie, allowing up to two mismatches. Sequence reads aligned to the mouse genome were imported into Partek Genomics Suite version 6.5 (Partek, St Louis, MO) for sequence read clustering, counting and annotation. The RefSeq transcript database was chosen as the annotation reference and subsequent data analyses were focused on sequence reads mapping to coding exons. The read counts of each known transcript were normalized to the length of the individual transcript and to the total mapped read counts in each sample and expressed as RPKM (reads per kilobase of exon per million mapped reads). Sequence reads mapped to different isoforms of individual genes were pooled together for subsequent comparative analyses.
Yang et al_miRNASeq.txt (mm9)
Yang et al_mRNASeq.txt (mm9)

Submission date

Jan 25, 2012

Last update date

May 15, 2019

Contact name

Kai-Chien Yang

Organization name

Washington University in St Louis

Department

Developmental Biology

Lab

Nerbonne