|
| Status |
Public on Mar 04, 2022 |
| Title |
IM_IC_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
3rd week LLC bearing lungs
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
sorted cell type: Ly6Chigh monocytes
treatment: IgG2b isotype control mAb
|
| Treatment protocol |
Four days after LLC-Fluc challenge, mice were treated with four intraperitoneal injections of 200µg mAb dissolved in 200 µl PBS given at 3-day intervals (days 4, 7, 10 and 13). Half was injected with rat IgG2b isotype control (IC) mAb (clone: LTF-2) , other half with rat IgG2b anti-PD-L1 mAb (clone: 10F9G2), both Biolegend.
|
| Growth protocol |
LLC-Fluc cells were maintained in DMEM+ i.e., Dulbecco’s-Modified-Eagle’s-Medium supplemented with 10% fetal bovine serum (FBS, TICO Europe), 100units/mL penicillin, 100 µg/mL streptomycin and 2 mM L-Glutamine (all Sigma-Aldrich) at 37°C, 5% CO2,21% O2 and 95% humidity level. Female 6-week-old C57BL/6 mice were injected intravenously with 5x1e5 LLC-Fluc cells dissolved in 200 µl phosphate-buffered-saline (PBS). All animals were handled according to the institutional guidelines, approved by the Ethical Committee for use of laboratory animals VUB (ECD: 18-214-1 and 18-281-9).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Myeloid immune cell types were sorted by FACS with a BD FACSAriaIII. 5x1e4 cells per condition were collected and stored in Trizol at -80°C prior to RNA extraction. RNA was isolated using the chloroform-isopropanol protocol. RNA recovery was quantified using fluorimetry (Qubit, LifeTechnologies) and qualified for purity and integrity using the nanophotometer (Implen) and Bioanalyzer Labchip (Agilent Technologies), resp. Due to low RNA concentrations, samples were pre-amplified using the nCounter Low-RNA-input kit (NanoString Technologies) with five amplification cycles.
|
| Label |
Biotin
|
| Label protocol |
After RNA was converted to cDNA and amplified using target-specific primers samples were mixed with a 3′ biotinylated capture probe and a 5′ reporter probe tagged with a fluorescent barcode from the nCounter Myeloid Innate Immunity Panel.
|
| |
|
| Hybridization protocol |
Probes and target transcripts were hybridized overnight at 65°C for 20 hours as described in the manufacturer’s protocol.
|
| Scan protocol |
Samples are loaded onto the nCounter Prep Station for further purification and immobilization onto the sample cartridge. After transfer to the Digital Analyzer (MAX Analysis System, Brightcore, VUB) absolute counts are quantified.
|
| Description |
/
|
| Data processing |
For quality control the nSolver_analysis_software_4.0 was used. Raw counts were processed in R and normalized using the RUVSeq method adjusted for NanoString analysis. The DESeq R-package was used for differential expression analysis.
|
| |
|
| Submission date |
Jan 26, 2021 |
| Last update date |
Mar 04, 2022 |
| Contact name |
Kirsten De Ridder |
| E-mail(s) |
kirsten.de.ridder@vub.be
|
| Organization name |
Vrije Universiteit Brussel
|
| Department |
Laboratory for Molecular and Cellular Therapy, Department of Biomedical Sciences
|
| Lab |
Kirsten De Ridder
|
| Street address |
Laarbeeklaan 103
|
| City |
Jette |
| State/province |
Brussel |
| ZIP/Postal code |
1090 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL25266 |
| Series (1) |
| GSE165517 |
Transcriptional analysis on the impact of anti-PD-L1 mAb on murine lung tumor sorted myeloid populations. |
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