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| Status |
Public on Apr 16, 2018 |
| Title |
CS3 BAL_IPF |
| Sample type |
SRA |
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| Source name |
lung bronchoalveolar lavage(BAL)
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| Organism |
Homo sapiens |
| Characteristics |
tissue: BAL macrophages
age: 61.1
Sex: male
disease: Idiopathic pulmonary fibrosis
smoker: Yes [Ex smoker]
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| Extracted molecule |
total RNA |
| Extraction protocol |
Fiberoptic bronchoscopy (FOB) and bronchoalveolar lavage(BAL) were carried out at first evaluation of IPF patients and control subjects. All FOBs and BALs were carried out following established procedures recommended by international guidelines, by instillation of 100 ml of room temperature normal saline solution per lobe in one to three lung lobes (right middle lobe, lingula or left lower lobe). Portions of BAL samples remaining after aliquots for diagnostic exams were taken, then used to generate the RNA sample bank, after informed consent was obtained. Lavage samples were maintained on ice and processed for RNA extraction within one hour of the endoscopic procedure. Total RNA from BAL cells (5x106) was obtained using the RNA/DNA/Protein Purification Kit (Norgen Biotech Corp., Thorold, ON, Canada). For the purpose of RNA analysis, study samples were selected based upon alveolar macrophage differential counts ( >80%), cell viability ( >95%), epithelial cells counts (<5%). Samples of BAL differential cell counts were done on Diff Quick (Bio-Optica, Milan, Italy) stained cytopreps (Cytospin Shandon 4, Thermo Scientific, Waltham, MA, USA). Samples were further selected after RNA quality assessment based upon RNA integrity (R.I.N.> 6.0) analyzed by Agilent 2100 Bioanalyzer with the Total RNA pico kit (Agilent Technologies, SantaClara CA).
For library preparation and high-throughput sequencing (Cofactor Genomics, Saint Louis, MO), cDNA was synthesized by random priming using the NuGEN Ovation FFPE RNA-seq system (NuGEN Technologies, San Carlos, CA), as recommended for clinical samples potentially affected by low RNA integrity [35], and fragmented to an average size of 200 bp using the Covaris S2 (Covaris, Woburn, MA). Fragmented cDNA was end repaired, A-tailed and ligated with barcoded Illumina adapters. Ligated DNA was treated with NuGEN mouse Insert Dependent Adaptor Cleavage (InDA-C) reagent to deplete cDNA corresponding to ribosomal RNA, and PCR amplified. Final library yield was measured using the Qubit High Sensitivity DNA assay (Thermo Fisher Scientific, Waltham, MA) and library size assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Raw sequence data in FASTQ format were assessed for Per Base Sequence Quality (Phred average score =33, ribosomal RNA content <2%) using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
NovoAlign (Novocraft, http://novocraft.com) was used to align reads to a set of transcript sequences and also to the reference genome (hg19).
Raw read counts were mapped on UCSC mRNA transcript human database based on the GRCh37/hg19 human genome version. NovoAlign parameters were set to allow multiple alignments to the transcriptome set to allow for isoforms, but only unique alignments to the genome.
The raw counts per transcript were then aggregated onto unique UCSC gene symbols of protein-coding mRNAs, to obtain a final table with non-normalized expression levels (counts) for 19723 unique gene symbol IDs. These data are contained in the file processed_data_matrix_counts.txt
Raw non-normalized data were fed into the R-Bioconductor tool edgeR to assemble differentially expressed gene lists.
Genome_build: hg19
Supplementary_files_format_and_content: raw read counts aggregated on UCSC mRNA transcripts database based on the GRCh37/hg19 human genome
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| Submission date |
Mar 23, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Ivan Arisi |
| E-mail(s) |
[email protected]
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| Phone |
+39-06-49255230
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| Organization name |
European Brain Research Institute
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| Department |
Bioinformatics Facility
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| Street address |
viale Regina Elena 295
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| City |
Roma |
| ZIP/Postal code |
00161 |
| Country |
Italy |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE79544 |
Idiopathic pulmonary fibrosis bronchoalveolar lavage cells RNA-seq indicates macrophage expression of pro-inflammatory M1/M2 activation concomitant with radical species metabolism inhibition |
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| Relations |
| BioSample |
SAMN04576541 |
| SRA |
SRX1657636 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2097615_CS3.sorted.bam.counts.txt.gz |
410.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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