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| Status |
Public on Dec 31, 2019 |
| Title |
B_CS_2 |
| Sample type |
SRA |
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| Source name |
Air Liquid Interface culture
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| Organism |
Homo sapiens |
| Characteristics |
donor: HC-2
lonza id: 501937
disease state: healthy
age: 42
Sex: F
ethnicity: Hispanic
smoker: N
treatment: CS
cigarettes per treatment: 4
dilution rate (l/min): 0.5
number of treatments a week: 3
cell type: Small Airway Epithelial Cells (SAECs)
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| Treatment protocol |
SAECs were exposed to whole cigarette smoke of K3R4F reference cigarettes (University of Kentucky, Lexington, KY, USA) using an automated cigarette smoking machine (In-Expose smoking robot, Scireq Montreal, QC, Canada) and the P.R.I.T. (Professional In-Vitro Technologies) ExpoCube in the experimental setup developed by Fraunhofer Institute for Toxicology and Experimental Medicine. Four cigarettes were smoked in parallel in compliance to ISO 3308, drawing every 15 seconds a puff from the sequentially smoked cigarettes (9 puffs per cigarette). The smoke was diluted with ambient air (dilution rate: 0.5 l/min). The cells were exposed to CS three times a week during the differentiation phase (28 days) beginning on day 0 (= day of air-lift). Readouts were performed 24 hours post last exposure.
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| Growth protocol |
Human Small Airway Epithelial Cells (SAEC, CC-2547, Lonza, Basel, Switzerland) were thawed and approximately one million cells were seeded into a T175 cell culture flask with 50 ml pre-warmed PneumaCult™-Ex Plus Medium (Stemcell Technologies, Vancouver, Canada). On the following day, the medium was exchanged, to remove residuals from the cryoprotectant cocktail. On the fourth day after seeding, the cells were sufficiently confluent (max. 80 %) to be harvested for seeding onto transwell inserts. The Transwells (#3460, Corning Life Sciences B.V., Amsterdam, the Netherlands) were coated with 300 µl rat tail collagen type 1 solution (Corning Life Sciences B.V., Amsterdam, the Netherlands, 30µg/ml in phosphate buffered saline) for 45 minutes at 37 °C. Collagen solution was aspirated and transwell inserts were washed with phosphate buffered saline. The basolateral compartments of the transwells were filled with 1.5 ml PneumaCult™-Ex Plus Medium. Dissociation of SAEC was performed using the Animal Component-Free Cell Dissociation Kit (Stemcell Technologies, Vancouver, Canada) according to the manufacturer’s instructions. Subsequently, 100.000 cells per insert were seeded in 300 µl on the transwell membrane. On the following day, apical and basolateral media was exchanged using PneumaCult™-Ex Plus Medium. On day three after the transfer to transwell inserts, when the cells were fully confluent, the apical and basolateral medium was removed and 1.5 ml Pneumacult-ALI-S Medium (Stemcell Technologies, Vancouver, Canada) was added basolaterally. The apical compartment was washed with phosphate buffered saline to remove residual growth factors of the media. The cells were from then on grown in air-liquid interface (day 0). Medium exchanges were performed three times a week. The cells were fully differentiated 28 days post air-lift. As stated by Lonza, the cells were isolated from donated human tissue after obtaining permission for their use in research applications by informed consent or legal authorization.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were individually extracted using the Ambion Magmax™-96 total RNA isolation kit (Life Sciences) according to the manufacturer’s instructions. Briefly, 5 mg of tissue was placed in the lysis solution and homogenised in Qiagen Tissuelyzer™ for a period of 30 sec. Nucleic acids were captured onto magnetic beads, washed and treated with DNase. Total RNA was then eluted in 50 μl elution buffer. RNA quality and concentration was measured using an RNA Pico chip on an Agilent Bioanalyzer.
The Sequencing library preparation has been done using 200 ng of total RNA input with the TrueSeq RNA Sample Prep Kit v2-Set B (RS-122-2002, Illumina Inc, San Diego, CA) producing a 275bp fragment including adapters in average size. Libraries have been clustered on the cBot Instrument from Illumina using the TruSeq SR Cluster Kit v3 - cBot - HS(GD-401-3001, Illumina Inc, San Diego, CA) sequencing was then performed on an Illumina HiSeq3000 instrument
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| Data processing |
Sequenced read quality was checked with FastQC v0.11.2
RNA-Seq reads from all samples were aligned to the genome using the STAR Aligner v2.5.2a11
Duplication rates of the RNA-Seq samples were computed with bamUtil v1.0.11 to mark duplicate reads and the dupRadar v1.4 Bioconductor R package for assessment
The gene expression profiles were quantified using Cufflinks software version 2.2.1 to get the Reads Per Kilobase of transcript per Million mapped reads (RPKM) as well as read counts from the feature counts software package
Genome_build: ensembl70
Supplementary_files_format_and_content: tab-delimited text file contains RPKM values for each sample
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| Submission date |
Jul 31, 2019 |
| Last update date |
Dec 31, 2019 |
| Contact name |
Karsten Quast |
| E-mail(s) |
karsten.quast@boehringer-ingelheim.com
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| Organization name |
Boehringer Ingelheim Pharma GmbH & Co. KG
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| Department |
Computational Biology
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| Street address |
Birkendorfer Str. 65
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| City |
Biberach |
| ZIP/Postal code |
88397 |
| Country |
Germany |
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| Platform ID |
GPL21290 |
| Series (1) |
| GSE135188 |
Intermittent exposure to whole cigarette smoke alters the differentiation of primary small airway epithelial cells in the air-liquid interface culture |
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| Relations |
| BioSample |
SAMN12407437 |
| SRA |
SRX6624169 |
| 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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