|
| Status |
Public on Dec 18, 2020 |
| Title |
d10 none 4 |
| Sample type |
RNA |
| |
|
| Source name |
CD4+ T cells primed in vivo, then exposed to different levels of persisting antigen
|
| Organism |
Mus musculus |
| Characteristics |
cells: sorted CD4+ AND T cells
days after transfer: 10
doxycycline [ug/ml] day -1 to 3: 100
doxycycline [ug/ml] day 3 to 10 or 30: 0
|
| Treatment protocol |
The recipients had been treated one day before the transfer with an anti-CD40 antibody to activate dendritic cells and with 100 µg/ml doycycline in the drinking water which induces the expression of the cDNA construct in dendritic cells. One group (none) was then withdrawn from doxycycline. We have seen before that the T cells in recipients undergoing this treatment will develop into Th1 memory cells (Obst et al., 2007). Another group (low) was switched to 10 µg/ml while a third (intermediate) was maintained on the 100 µg/ml dose. In addition, another strain of mice (cMCC) were used as recipients that expressed an invariant chain/MCC construct that is constitutively expressed in MHC-II+ cells at high levels (high). The adoptively transferred AND TCR tg T cells were sorted twice on day 10 as CD4+CD90.2+CD45.1+ cells directly into Trizol LS , mixed and stored at -80°C for processing. - A second series of sorts was done 30 days after transfer. Instead of the cMCC recipients, in which the AND T cells undergo apoptosis by then, iMCC animals were used which were taken off doxycycline on day 10 (off). Naive control AND T cells were transferred 3 days before the sort into non-transgenic recipients.
|
| Growth protocol |
AND TCR transgenic CD45.1+ Rag2o/o CD4+ T cells were adoptively transferred into iMCC recipients carrying two transgenes that allow for the dynamic regulation of the expression of an invariant chain cDNA which carries a moth cytochrome c (MCC) epitope instead of the CLIP sequence.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The Trizol samples were thawed on ice, 300 µl chloroforn was added, shaken, incubated for 3 minutes at room temperature and spun at 12,000 x g and 4°C. The aqueous phase was transferred to new tubes, 1 volume of isopropanol and 1 µl of linear polyachrylamide stock solution (25 µg/µl) was added. The samples were precipitated for 16 hours at -20°C and centrifuged for 10 minutes at 12,000 x g and 4°C. Supernatants were removed and RNA pellets washed with 70% ethanol. RNA pellets were resuspended in 100 µl RNAse-free water and purified with NucleoSpin RNA Clean-up XS columns as per manufacturer's instructions, from which the RNA was eluted with 12 µl water and stored at -80°C. RNAs were quantified with an Agilent 2100 Bioanalyzer. All samples had RNA integrity values > 8 and were use for amplification and microarray hybridizations.
|
| Label |
biotin
|
| Label protocol |
Total RNA (0.1-30 ng) was amplified and labeled using the Ovation Pico WTA System V2 in combination with the Encore Biotin Module (Nugen).
|
| |
|
| Hybridization protocol |
Amplified cDNA was hybridized on Affymetrix Mouse Gene ST 2.0 arrays containing about 35,000 probe sets.
|
| Scan protocol |
Staining and scanning using a GeneChip Scanner 3000 7G was done according to the Affymetrix expression protocol including minor modifications as suggested in the Encore Biotin protocol.
|
| Data processing |
R/Bioconductor, rma normalization, default parameters
|
| |
|
| Submission date |
Jan 15, 2020 |
| Last update date |
Dec 18, 2020 |
| Contact name |
Tobias Straub |
| E-mail(s) |
tstraub@med.uni-muenchen.de
|
| Organization name |
LMU Munich
|
| Department |
Biomedical Center, Bioinformatics
|
| Street address |
Großhadener Str. 9
|
| City |
Martinsried |
| ZIP/Postal code |
82152 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16570 |
| Series (2) |
| GSE143733 |
Gene expression of CD4+ T cells in response to antigen persisting at different doses for different times |
| GSE143739 |
Gene expression of CD4+ T cells |
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