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Sample GSM1579214

Query DataSets for GSM1579214

Status

Public on Jan 07, 2018

Title

Pool healthy and asthma (mRNA)

Sample type

SRA

Source name

lung

Organism

Homo sapiens

Characteristics

tissue: lung
cell type: bronchial smooth muscle (BSM) cells
disease state: pool healthy and asthma

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

polyA 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).
For small RNA profiling, 1 µg of total RNA was used for library preparation according to the Illumina TruSeq small RNA sample preparation protocol. Sized microRNA libraries were gel purified and sequenced on a Illumina HiSeq1500 sequencer. For polyA RNA sequencing, an equimolar RNA pool was prepared from all 14 RNA samples extracted from both asthmatic and normal primary BSM cells. 1 µg of total RNA was used for library preparation according to the Illumina TruSeq stranded mRNA sample preparation protocol. Resulting mRNA library was sequenced on a Illumina NextSeq sequencer.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

processed data file: mRNA-Seq_read_count.txt

Data processing

Basecalls was performed using CASAVA version 1.8
iMir was used to identify members of the following sncRNA families: microRNAs, tRNAs or rRNAs, piRNAs RFam and tiRNAs with Minimum Read Count of 3, Minimum Read Length of 17, minReadlengthTrans of 32 and maxReadLength of 32. Reads were normalized using reads per million method (RPM). ToAdaptor sequences and low-quality terminal nucleotide positions within the reads were removed using Trimmomatic software
Sequenced reads for RNA-Seq libraries were aligned to human reference genome with TopHat (v 2.0.10) using the -G (GTF file of Ensembl release 75) option. Further, the aligned reads are used to quantify mRNA expression by using the HTSeq-count (v 0.6.1) with hg19 GTF (Ensembl release 75).The pre-processed reads were mapped to the human genome, using the software Bowtie2.
Genome_build: hg 19
Supplementary_files_format_and_content: all_sncRNA_RPM_normalized.txt file includes RPM normalized sncRNA expression values for each Sample
Supplementary_files_format_and_content: mRNA-Seq_read_count.txt file contains read counts for genes that have read count ≥10

Submission date

Jan 07, 2015

Last update date

May 15, 2019

Contact name

Elena Alexandrova

E-mail(s)

ealexandrova@unisa.it

Phone

+393661595308

Organization name

University of Salerno