|
| Status |
Public on Sep 04, 2020 |
| Title |
gall bladder epithelium_early organoids P4_vs_differentiated organoids P4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
differentiated organoids P4
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: gall bladder
cell type: epithelium
treatment: not infected, harvested day 14
bacteria_strain: none
culture type: organoids
replicate: P4
|
| Treatment protocol |
Organoids were harvested 4 or 14 days (for early and differentiated organoids, respectively) after seeding. For infection experiments, 2D primary cells infected with /Salmonella/ paratyphi A were harvested 5 days post infection, FACS sorted based on the /Salmonella/ mCherry fluorescence to get 3x10^5 infected cells for RNA extraction.
|
| Growth protocol |
Human gall bladder epithelial cells were derived from patients that underwent cholecystectomy, stored in ice-cold PBS for up to 2 hours and the tissue was washed with PBS from the residual bile and mucus and it was incubated at 37°C with the mucosal side facing a solution of 0.2% collagenase type IV. The mucosa was abraded thoroughly with the end of a glass microscope slide held at an angle of 45°every 5 minutes, for 4 times. The isolated cells were passed through a cell strainer with 70μm pores, spun down and 1 to 3 million cells were resuspended in a drop of 50μl matrigel. The polymerized matrigel drop was then supplemented with a medium based on Advanced/DMEM F12 (Invitrogen). The medium was changed twice a week. Every 7 to 10 days the organoids were spit with a ratio 1 to 3-4 by treating with Trypsin, then passing them 10 times through a heat-narrowed Pasteur pipet.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by the TRIzol Reagent RNA preparation method (Invitrogen, Karlsruhe, Germany).
|
| Label |
Cy3
|
| Label protocol |
RNA was labeled with the Low Input Quick Amp Labeling Kit (Agilent Technologies) according to manufacturer’s instructions.
|
| |
|
| Channel 2 |
| Source name |
early organoids P4
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: gall bladder
cell type: epithelium
treatment: not infected, harvested day 4
bacteria_strain: none
culture type: organoids
replicate: P4
|
| Treatment protocol |
Organoids were harvested 4 or 14 days (for early and differentiated organoids, respectively) after seeding. For infection experiments, 2D primary cells infected with /Salmonella/ paratyphi A were harvested 5 days post infection, FACS sorted based on the /Salmonella/ mCherry fluorescence to get 3x10^5 infected cells for RNA extraction.
|
| Growth protocol |
Human gall bladder epithelial cells were derived from patients that underwent cholecystectomy, stored in ice-cold PBS for up to 2 hours and the tissue was washed with PBS from the residual bile and mucus and it was incubated at 37°C with the mucosal side facing a solution of 0.2% collagenase type IV. The mucosa was abraded thoroughly with the end of a glass microscope slide held at an angle of 45°every 5 minutes, for 4 times. The isolated cells were passed through a cell strainer with 70μm pores, spun down and 1 to 3 million cells were resuspended in a drop of 50μl matrigel. The polymerized matrigel drop was then supplemented with a medium based on Advanced/DMEM F12 (Invitrogen). The medium was changed twice a week. Every 7 to 10 days the organoids were spit with a ratio 1 to 3-4 by treating with Trypsin, then passing them 10 times through a heat-narrowed Pasteur pipet.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by the TRIzol Reagent RNA preparation method (Invitrogen, Karlsruhe, Germany).
|
| Label |
Cy5
|
| Label protocol |
RNA was labeled with the Low Input Quick Amp Labeling Kit (Agilent Technologies) according to manufacturer’s instructions.
|
| |
|
| |
| Hybridization protocol |
After precipitation, purification and quantification, labeled samples were hybridized according to the supplier's protocol (Agilent Technologies).
|
| Scan protocol |
Scanning of microarrays was performed with 3µm resolution using a G2565CA high resolution laser microarray scanner (Agilent Technologies).
|
| Data processing |
Microarray image data were extracted and analyzed with the Image Analysis / Feature Extraction software G2567AA (Version A.10.10.1, Agilent Technologies) using default settings and the GE1_1105_Oct12 extraction protocol. The extracted dual-color raw data txt files were further analyzed using R and the associated BioConductor package limma. Agilent FE software processed green and red signals were extracted and within-array normalized using method LOESS followed by averaging of duplicated probes was applied.
|
| |
|
| Submission date |
Jun 29, 2017 |
| Last update date |
Sep 06, 2020 |
| Contact name |
Thomas F Meyer |
| E-mail(s) |
tfm@mpiib-berlin.mpg.de
|
| Phone |
03028 460 404
|
| Organization name |
Max Planck Institute for Infection Biology
|
| Department |
Molecular Biology
|
| Street address |
Chariteplatz 1
|
| City |
Berlin |
| ZIP/Postal code |
10117 |
| Country |
Germany |
| |
|
| Platform ID |
GPL21272 |
| Series (1) |
| GSE100656 |
Gene expression profiles of primary human gall bladder epithelial cells with and without infection with S.paratyphi WT and ∆cdtB mutant for 2 and 7 days |
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