|
| Status |
Public on Jan 31, 2019 |
| Title |
254890810729_2_2: D2 long term acute vs. D2 naive (cured) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
D2 naive - cured - Ctr infection
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: fallopial tube
donor: organoid donor 2
progression: long term acute
|
| Treatment protocol |
For infection with Chlamydia trachomatis D (CtrD) 2 wells of organoids were released from MatrigelTM with cold DPBS, pooled and mechanically disrupted by passing them through a 26xG needle. The fragmented organoids were divided equally into 2 tubes for infection and mock control. After centrifugation (300xg, 5min) and removal of the supernatant, 10µl CtrD (from a frozen stock with 108 IFU/mL as determined by titration on HeLa cells) were added to one tube of the pelleted organoids. The suspension was mixed by pipetting and incubated for 15min at 35°C followed by 5min on ice. The mock control was treated the same, but without addition of CtrD. Finally, 50µL MatrigelTM was added to each tube, the organoids seeded and placed in a humidified incubator at 5% CO2 and 35°C. Splitting of organoids was done every 3-4 weeks. RNA samples were taken at 72h p.i. and 1M p.i. At 4 months p.i. the infection was cured by applying a mixture of Penicillin (100 U/mL) and Streptomycin (100 µg/mL) to the organoid culture for one week.
After curing, a re-infection was carried out identical to the first infection with mechanical splitting, adding of CtrD to one half of the organoids and mock treatment of the other half. Also, the control organoids that have not been infected with CtrD before were infected and mock treated again. 72h post seeding RNA samples were taken of the now 2x infected (chronic cured acute), chronic infected (chronic cured), acute infected (longterm acute) and the never infected organoids
|
| Growth protocol |
Epithelial progenitor isolation from fallopian tube samples and organoid culture was done as previously described (Nat Commun. 2015 Dec 8;6:8989. doi: 10.1038/ncomms9989.)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by the Trizol (Invitrogen) method following the manufacturer´s protocol.
|
| Label |
Cy3
|
| Label protocol |
RNA labeling was performed with the Quick Amp RNA Fluorescent Linear Amplification Kit (Agilent Technologies) according supplier's instructions.
|
| |
|
| Channel 2 |
| Source name |
D2 naive - cured
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: fallopial tube
donor: organoid donor 2
progression: naive (cured)
|
| Treatment protocol |
For infection with Chlamydia trachomatis D (CtrD) 2 wells of organoids were released from MatrigelTM with cold DPBS, pooled and mechanically disrupted by passing them through a 26xG needle. The fragmented organoids were divided equally into 2 tubes for infection and mock control. After centrifugation (300xg, 5min) and removal of the supernatant, 10µl CtrD (from a frozen stock with 108 IFU/mL as determined by titration on HeLa cells) were added to one tube of the pelleted organoids. The suspension was mixed by pipetting and incubated for 15min at 35°C followed by 5min on ice. The mock control was treated the same, but without addition of CtrD. Finally, 50µL MatrigelTM was added to each tube, the organoids seeded and placed in a humidified incubator at 5% CO2 and 35°C. Splitting of organoids was done every 3-4 weeks. RNA samples were taken at 72h p.i. and 1M p.i. At 4 months p.i. the infection was cured by applying a mixture of Penicillin (100 U/mL) and Streptomycin (100 µg/mL) to the organoid culture for one week.
After curing, a re-infection was carried out identical to the first infection with mechanical splitting, adding of CtrD to one half of the organoids and mock treatment of the other half. Also, the control organoids that have not been infected with CtrD before were infected and mock treated again. 72h post seeding RNA samples were taken of the now 2x infected (chronic cured acute), chronic infected (chronic cured), acute infected (longterm acute) and the never infected organoids
|
| Growth protocol |
Epithelial progenitor isolation from fallopian tube samples and organoid culture was done as previously described (Nat Commun. 2015 Dec 8;6:8989. doi: 10.1038/ncomms9989.)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by the Trizol (Invitrogen) method following the manufacturer´s protocol.
|
| Label |
Cy5
|
| Label protocol |
RNA labeling was performed with the Quick Amp RNA Fluorescent Linear Amplification Kit (Agilent Technologies) according supplier's instructions.
|
| |
|
| |
| Hybridization protocol |
Whole human genome microarrays were done according to the supplier’s protocol (Agilent Technologies) using the SSPE wash protocol
|
| Scan protocol |
Scanning of microarrays was performed with either 5 µm resolution and XDR setting for 4x44K arrays or 3 µm resolution for 8x60K arrays using a G2505C DNA microarray laser scanner (Agilent Technologies)
|
| Description |
(-) polarity
254890810729_2_2
|
| Data processing |
Raw microarray image data were analyzed with the Image Analysis / Feature Extraction software G2567AA (Version A.11.5.1.1, Agilent). The extracted files were analyzed with the Rosetta Resolver Biosoftware (Rosetta Biosoftware) and with Bioconductor Gene Spring 12.6.1 software (Agilent Technologies).
|
| |
|
| Submission date |
Dec 05, 2017 |
| Last update date |
Jan 31, 2019 |
| Contact name |
Hans-Joachim Mollenkopf |
| E-mail(s) |
mollenkopf@mpiib-berlin.mpg.de
|
| Phone |
+49 30 28460 482
|
| Organization name |
Max-Planck-Institute for Infection Biology
|
| Lab |
Microarray/Genomics Core Facility
|
| Street address |
Charitéplatz 1
|
| City |
Berlin |
| ZIP/Postal code |
10117 |
| Country |
Germany |
| |
|
| Platform ID |
GPL21272 |
| Series (1) |
| GSE107712 |
Chronic Chlamydia infection in fallopian tube organoids alters epithelial differentiation and accelerate methylation changes |
|
