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Sample GSM1556635 Query DataSets for GSM1556635
Status Public on Feb 17, 2016
Title Control_706
Sample type SRA
 
Source name lung
Organism Homo sapiens
Characteristics tissue: lung
sample type: bronchial smooth muscle cells
disease state: healthy
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.
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection CAGE
Instrument model Illumina HiSeq 1500
 
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 …
 
Submission date Dec 01, 2014
Last update date May 15, 2019
Contact name Elena Alexandrova
E-mail(s) ealexandrova@unisa.it
Phone +393661595308
Organization name University of Salerno
Department Faculty of Medicine and Surgery
Lab Laboratory of Molecular Medicine and Genomics
Street address Viale Stazione, 22
City Portici
State/province Napoli
ZIP/Postal code 80055
Country Italy
 
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.
Relations
BioSample SAMN03247197
SRA SRX791654

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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