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| Status |
Public on Feb 17, 2016 |
| Title |
Control_685 |
| Sample type |
SRA |
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| Source name |
lung
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| Organism |
Homo sapiens |
| Characteristics |
tissue: lung
sample type: bronchial smooth muscle cells
disease state: healthy
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| Growth protocol |
BSM cells were established and grown in RPMI 1640 (Lonza, Basel, Switzerland) supplemented with 5% fetal calf serum (FCS), 8 mM L-glutamine, 20 mM hydroxyethyl piperazine ethane sulfonic acid and 1% modified Eagle’s medium vitamin mix (Gibco, Paisley, UK). Neither antibiotics nor antimycotics were added at any time.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolation was performed with mirVana miRNA isolation kit (Ambion, Life Science, Zug, Switzerland). RNA concentration of each sample was evaluated with a ND-1000 spectrophotometer (NanoDrop) and its quality was analysed with the Agilent 2100 Bioanalyzer using Agilent RNA 6000 nano kit (Agilent Technologies, Milano, Italy).
5 µg of total RNA was reverse transcribed with RT random N15 primer using PrimeScript Reverse Transcriptase in the presence of 0.132 M trehalose and 0.66 M sorbitol. The 5’ RNA caps were than oxidated with NaIO4, biotinilated and treated with RNAseI ribonuclease in order to cleave single-stranded RNA regions that were not protected by cDNA. Next, 5’-completed cDNA-RNA hybrids were cap-trapped, cDNA molecules were released and a specific linker (5'-linker), containing a 3-bp barcode sequence and the type III restriction-modification enzyme EcoP15I was ligated to the single-strand cDNA. The priming of the second strand was made with specific biotinilated primer. After second strand synthesis and cleavage with EcoP15I, another linker (3'-linker) was ligated. Purified cDNA was amplified with 1 μM each, forward and reverse primers with 15 PCR cycles. PCR products were purified and concentration was adjusted to 10 nM.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
CAGE |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
Basecalls was performed using CASAVA version 1.8
Adaptor sequences and low-quality terminal nucleotide positions within the reads were removed using Trimmomatic software
The pre-processed reads were mapped to the human genome, version HG 19, using the software Bowtie2
The mapped reads were attributed to known human genes using the published Ensembl human genome annotation Vers. 75
Differential gene expression data normalized on the overall number of mapped sequenced reads were obtained using DESeq software
Genome_build: hg 19
Supplementary_files_format_and_content: .txt file include DESeq normalized gene expression values for each Sample …
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| Submission date |
Dec 01, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Elena Alexandrova |
| E-mail(s) |
ealexandrova@unisa.it
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| Phone |
+393661595308
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| Organization name |
University of Salerno
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| Department |
Faculty of Medicine and Surgery
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| Lab |
Laboratory of Molecular Medicine and Genomics
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| Street address |
Viale Stazione, 22
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| City |
Portici |
| State/province |
Napoli |
| ZIP/Postal code |
80055 |
| Country |
Italy |
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| Platform ID |
GPL18460 |
| Series (1) |
| GSE63744 |
Large-scale profiling of intracellular signalling pathway activation reveals major distinctions between airway smooth muscle cells of asthmatics and non-asthmatics. |
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| Relations |
| BioSample |
SAMN03247199 |
| SRA |
SRX791651 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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