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Sample GSM866644 Query DataSets for GSM866644
Status Public on May 01, 2013
Title WT Sham 6 LV miRNA Seq
Sample type SRA
Source name WT Sham 6 LV RNA
Organism Mus musculus
Characteristics genotype: WT
strain: FVB
tissue: heart left ventricle
treatment: Sham
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
Street address 660 South Euclid Avenue Box 8103
City St Louis
State/province MO
ZIP/Postal code 63110-1093
Country USA
Platform ID GPL13112
Series (1)
GSE35350 Combined miRNA and mRNA Sequencing Identifies Protective Transcriptional Signature of Enhanced PI3Kalpha Signaling in Cardiac Hypertrophy
SRA SRX117892
BioSample SAMN00779122

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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