|
| Status |
Public on May 29, 2024 |
| Title |
Human lung tissue cells uninfected control |
| Sample type |
SRA |
| |
|
| Source name |
Lung
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Lung
cell type: Primary
genotype: COPD
treatment: uninfected
|
| Treatment protocol |
AfterAfter 12-16 h of washing, tissues were infected with the respective pathogen.
|
| Growth protocol |
Bronchial lung tissue explanted from patients with terminal COPD for clinical reasons was obtained from the Institute of Pathology, Hannover Medical School. The tissue was further dissected into small pieces with an average size of approx. 27 mm3 (3x3x3 mm) and an average weight of approx. 30 mg. These pieces (HLTEs) were cultured in RPMI medium without any supplement. Tissue pieces were cultured overnight in a humidified tissue culture incubator at 37°C, 5% CO2; this step was termed overnight washing.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
After 24 hpi, tissues were collected and processed to prepare a single-cell suspension for flow cytometry and cell sorting. Briefly, lung tissues were chopped in digestion buffer (1 mg/ml collagenase D (Roche, 10269638001), 2.0 U/ml Dispase II (Roche, 04942078001) and 0.1 mg/ml DNase I (DNase I, D4527) in Hepes-buffer) with surgical scissors and homogenized with an Ultra-Turrax® dispersing tool (T10, IKA-Werke GmbH, Staufen, Germany) for 40-60 sec. The homogenate was incubated for 30 minutes at 37°C with mild agitation and then passed through a 30G syringe. The resulting single-cell suspension was filtered through a 40 µm nylon cell strainer and erythrocytes were lysed using ACK (Ammonium-Chloride-Potassium) lysing buffer. Cells were counted using an automated cell counter (Scepter, Millipore, PHCC00000).
Single-cell 3’ RNA-Seq libraries were prepared using Chromium Single Cell V3 Reagent Kit and Controller (10x Genomics) as described in the user guide. In brief, cells (n=4500) for a targeted recovery of 3500 along with gel beads, master mix and partitioning oil were loaded in designated wells on a chromium chip. The chip was placed in a Chromium Controller and run for Gel Bead-in-Emulsion (GEMs) preparation. After completion of the run, GEMs were run in PCR tubes for RT incubation in a thermal cycler. Post GEM-RT incubation, cDNA was cleaned up and amplified. cDNA was washed and quality was evaluated. Gene expression libraries were constructed and then libraries were assessed for quality (TapeStation 4200, Agilent). Libraries were sequenced by NextSeq550 using the NextSeq 500 High Output Kit v2.5 (1x75 cycles 400M cluster).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
Single cell transcriptome from total 2964 human lung tissue untreated. Cell were uninfected and untreated.
|
| Data processing |
Cell Ranger 1.3 (http://10xgenomics.com) was used to process Chromium single cell 3’ RNA-seq output
cellranger count aligned to the pre-built human reference genome (hg19, GRCh38)
Raw counts were normalized by the global-scaling normalization method “LogNormalize”.
Principal component analysis score was used to determine the ‘dimensionality’ of all cells.
Scatter plots were obtained using the UMAP method. Cell clusters were identified as different cell populations based on the expression of recently published canonical markers
Assembly: human reference genome (hg19, GRCh38) and Influenza A virus
Supplementary files format and content: filtered_feature_bc_matrix, tsv and mtx files
Supplementary files format and content: Seurat object
|
| |
|
| Submission date |
May 29, 2024 |
| Last update date |
May 29, 2024 |
| Contact name |
Frank Pessler |
| E-mail(s) |
frank.pessler@helmholtz-hzi.de
|
| Phone |
0511-220027-167
|
| Organization name |
TWINCORE Centre for Experimental and Clinical Infection Reearch
|
| Department |
Experimental Infection Research
|
| Lab |
Research Group Biomarkers for Infectious Diseases
|
| Street address |
Feodor-Lynen-Str. 7
|
| City |
Hannover |
| State/province |
Lower Saxony |
| ZIP/Postal code |
30625 |
| Country |
Germany |
| |
|
| Platform ID |
GPL21697 |
| Series (1) |
| GSE268542 |
10x single-cell RNAseq profiling of influenza Virus infected Human Lung Tissue Explants (HLTE) |
|
| Relations |
| BioSample |
SAMN41579553 |
| SRA |
SRX24732576 |
