 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jan 08, 2024 |
| Title |
Wood smoke, bronchial, chronic bronchitis-like, replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
bronchial
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: bronchial
disease state: chronic bronchitis-like
treatment: Wood smoke
|
| Treatment protocol |
As described in the paper. Briefly, the wood smoke was diluted in the ratio 1:20 with clean filtered room air. The exposure duration of 10 minutes (min) was decided based on the findings of a pilot dose response experiment (5,10, and 15 min exposure duration) assessing cytotoxicity (lactate dehydrogenase assay in basal media) and transcript expression of tumor necrosis factor (TNF; proinflammatory marker) and superoxide dismutase 3, extracellular (SOD3; oxidative stress marker). The medium exposure dose was then chosen accordingly. None of the doses were cytotoxic.
The bro-ALI, bro-ALI-CB, and alv-ALI models were developed in transwell inserts in 12-well plates as previously described. After change of cell medium, the plates were placed in a 3L desiccator glass jar maintained at 37°C and 60% humidity and allowed to equilibrate for 5 min. The volume of the desiccator represents the functional residual capacity of a healthy adult human lung35. As the temperature of the electric smoker reached 80°C, the pump was switched on to suck wood smoke at a flow rate of 60 ml per min along with filtered air at a flow rate of 1.2 L per minute into the desiccator to obtain the 1:20 dilution.
The lung models were exposed to wood smoke or filtered clean air (Sham) for 10 min, where after they were transferred to a cell incubator (37°C, 60% humidity and 5% CO2) for 3 hours (h) until next exposure session on the same day. Two exposure sessions were performed on days 1 and 2; and one exposure on day 3 (5 exposures in total. Following completion of repeated exposures each day, the lung models were incubated for 24h (since start of exposure on previous day). Cell culture medium was collected (and stored at -80°C) and replaced with fresh ALI medium 24 h after the first exposure each day and prior to the start of the exposure on the following day. Cell inserts were collected at the completion of 72 h since start of first exposure on day 1. The exposure regime did not induce cytotoxicity in any of bro-ALI, bro-ALI-CB, or alv-ALI models and therefore further molecular analysis was carried out. Cytotoxicity was assessed by lactate dehydrogenase (LDH; cat# 88953; Thermo Fisher scientific) for the pilot studies and propidium iodide staining (cat#556463; BD bioscience) for the repeated exposure studies. Sham (clean air) exposed samples under identical conditions served as control.
|
| Growth protocol |
As described in the paper. Briefly, the bro-ALI and bro-ALI-CB models were developed using PBEC harvested from macroscopically normal bronchial tissue obtained from one donor in connection with lobectomy following written and informed consent as described in previous studies. The protocol was approved by the Swedish Ethical Review Authority (Institutional ethic committee reference number: 99-357; approved on 10th January 2000). All methods were performed in accordance with the relevant guidelines and regulations. The bro-ALI and bro-ALI-CB models were developed according to the procedure described previously [PMID: 28107509; PMID: 31462114]. Interleukin 13 (IL13;1 ng/ml) was added to the basal medium of each insert from the 1st day of PBEC in ALI condition to develop the bro-ALI-CB models. The detailed protocol and cellular differentiation (club cells, goblet cells, basal cells, ciliated cells, etc.) of the PBEC-ALI and PBEC-ALI-CB model development have been described in detail previously (PMID: 28107509). The models have been used in several other studies.
The alv-ALI model was developed using NCI-H441 (ATCC HTB-174; derived from the pericardial fluid of a patient with papillary adenocarcinoma of the lung) cell line. The detailed protocol and model characteristics have been described in detail previously and used in several recent studies [PMID: 33235237, PMID: 34960806, PMID: 36180488]. The NCI-H441 cells, representative of human type II pneumocytes, express constitutively the mRNA and protein of the major surfactant apo-protein. NCI-H441 cells were co-cultured with HULEC-5a (ATCC CRL-3244) representative of human lung microvascular endothelial cells for this purpose. The alv-ALI model characterization included light microscopy, confocal microscopy, transmission electron microscopy, and transepithelial electrical resistance measurement. Morphological characterization of the alv-ALI model demonstrated the presence of tight junction protein 1, lamellar bodies, surfactant protein C, microvilli, lipid bodies, desmosome, and tight junctions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNeasy Micro (QIAGEN)
UPX 3’ RNA sequencing technology using Qiagen Genomic Services, Germany
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
BMS_CB_17
|
| Data processing |
The “Demultiplex QIAseq UPX 3’ reads” tool of the CLC Genomics Workbench 21.0.4 was used to demultiplex the raw sequencing reads according to the sample indices. The “Quantify QIAseq UPX 3’ workflow” was used to process the demultiplexed sequencing reads with default settings. In short, the reads are annotated with their UMI and are then trimmed for poly(A) and adapter sequences, minimum reads length (15 nucleotides), read quality, and ambiguous nucleotides (maximum of 2). They are then deduplicated using their UMI. Reads are grouped into UMI groups when they (1) start at the same position based on the end of the read to which the UMI is ligated (i.e., Read2 for paired data), (2) are from the same strand, and (3) have identical UMIs. Groups that contain only one read (singletons) are merged into non-singleton groups if the singleton’s UMI can be converted to a UMI of a non-singleton group by introducing an SNP (the biggest group is chosen). The reads were then mapped to the Human genome GRCh38 The ‘Empirical analysis of DGE’ algorithm of the CLC Genomics Workbench 21.0.4 was used for differential expression analysis with default settings. It is an implementation of the ‘Exact Test’ for two-group comparisons developed by Robinson and Smyth, 2008 (Small-sample estimation of negative binomial dispersion, with applications to SAGE data) and incorporated in the EdgeR Bioconductor package (Robinson et al., 2010, edgeR: a Bioconductor package for differential expression analysis of digital gene expression data). Genome_build: Human genome GRCh38 Supplementary_files_format_and_content: tab-delimited text files include normalized CPM values for each sample
Assembly: GRCh38
Supplementary files format and content: tab-delimited text files include normalized CPM values for each sample
|
| |
|
| Submission date |
Jul 07, 2023 |
| Last update date |
Jan 09, 2024 |
| Contact name |
Johannes Beckers |
| E-mail(s) |
johannes.beckers@helmholtz-munich.de
|
| Organization name |
Helmholtz Zentrum Muenchen
|
| Department |
Institute of Experimental Genetics
|
| Street address |
Ingolstaedter Landstr. 1
|
| City |
Neuherberg |
| ZIP/Postal code |
85764 |
| Country |
Germany |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE236857 |
Assessment of wood smoke induced pulmonary toxicity using bronchial, chronic bronchitis-like, and alveolar lung mucosa models at air-liquid interface |
|
| Relations |
| BioSample |
SAMN36366910 |
| SRA |
SRX20942957 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7578112_WS_CB_17.txt.gz |
169.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
|
|
|
|
 |
