|
| Status |
Public on May 26, 2020 |
| Title |
lungNHBE_2: hBEpC_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
primary human bronchial epithelial cells_rep2
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Bronchi
|
| Treatment protocol |
Approximately 100 organoids were infected with 7.0×10^4 TCID50 of SARS-CoV-2 in 24 well plate containing 500 uL differentiation medium. The one-half of differentiation medium containing SARS-CoV-2 was replaced with fresh differentiation medium every day. After 5 days from the infection, the hBO and their supernatant were collected. In the drug treatment experiment, the infected hBO were cultured with the differentiation medium containing 10 μM camostat (Sigma-Aldrich) for 5 days.
|
| Growth protocol |
Normal human bronchial epithelial cells (hBEpC, Lonza) were suspended in 10 mg/ml cold Matrigel growth factor reduced (GFR) basement membrane matrix. 50 μL drops of cell suspension were solidify on pre-warmed Nunc cell-culture treated multidishes (24 well plate) at 37°C for 10 min, and then 500 μL of the expansion medium was added to each well. The expansion medium was changed every 2 days. HBO were passaged every 10-12 days. To passage the hBO, organoids were suspended in 1 mL 0.5 mM EDTA/PBS (Nacalai tesque) and mechanically sheared using P1000 pipette tip, and 2 mL TrypLE Select (Thermo Fisher Scientific) was added to the suspension. After incubating for 5 min at room temperature, organoids were mechanically sheared using P1000 pipette tip. 7 mL of the expansion medium was added, and the organoid suspension tubes were centrifuged at 400 rpm. Organoid fragments were re-suspended in the cold expansion medium and seeded as above. The hBO were passaged every 10 days. To maturate the hBO, the expanded hBO were cultured with the differentiation medium for 5 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from hBO and A549 cells using ISOGENE II (NIPPON GENE).
Library preparation was performed using a NEBNext Ultra II Directional RNA Library Prep Kit for an Illumina NextSeq500 and a TruSeq stranded mRNA sample prep kit for NovaSeq6000 platform according to the manufacturer’s instructions.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
primary human bronchial epithelial cells_rep2
|
| Data processing |
Sequencing was performed on an Illumina NextSeq500 or NovaSeq6000 platform in a 152 or 101-base single-end mode, respectively.
Illumina RTA 2.4.11 and Casava1.8.2 softwares were used for basecalling for an Illumina NextSeq500 or NovaSeq6000 platform, respectively. .
Fastq files were generated using bcl2fastq2.
Adapter sequences were trimmed from raw reads by cutadapt ver 2.7 and then trimmed reads were mapped to the human reference genome sequences (hg19) using HISAT2 v2.1.0.
The raw counts were calculated using featureCounts v 2.0.0.
Genome_build: hg19
|
| |
|
| Submission date |
May 19, 2020 |
| Last update date |
Jun 10, 2020 |
| Contact name |
Daisuke Okuzaki |
| E-mail(s) |
[email protected]
|
| Phone |
+81-6-6879-4935
|
| Organization name |
Osaka univ.
|
| Department |
Immunology Frontier Research Center
|
| Lab |
Human Immunology （Single Cell Genomics)
|
| Street address |
Yamadaoka 3-1
|
| City |
Suita |
| State/province |
Osaka |
| ZIP/Postal code |
565-0871 |
| Country |
Japan |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE150819 |
Generation of human bronchial organoids for SARS-CoV-2 research |
|
| Relations |
| BioSample |
SAMN14970479 |
| SRA |
SRX8362305 |
