|
| Status |
Public on Sep 25, 2023 |
| Title |
BALF-MP-WTM-CFL103 |
| Sample type |
RNA |
| |
|
| Source name |
Bronchoalveolar lavage fluids
|
| Organism |
Macaca fascicularis |
| Characteristics |
cell type: CD64+ macrophages
viral status: >221 days post infection with the wild type Wuhan strain
|
| Growth protocol |
Macrophages were isolated from BALF samples using a positive selection strategy. We employed the anti-CD64 PE-conjugated monoclonal antibody (clone 10.1, BD Bioscience, USA) for this purpose.To reveal the isolated macrophages, anti-PE microbeads from Miltenyi Biotec (USA) were utilized. These macrophages were cultured for 8h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After the incubation, the cells were resuspended in 30µl of RLT buffer with beta-mercaptoethanol (Qiagen) following the Nanostring protocols recommendation for cell Lysate (MAN-10051-05).
|
| Label |
n/a
|
| Label protocol |
The system uses molecular “barcodes” and single molecule imaging to detect and count hundreds of unique transcripts in a single reaction.
|
| |
|
| Hybridization protocol |
Seventy microliters of hybridization buffer was added to the reporter code set to make a master mixture. 2,1µl of proteinase K were added. Eight microliters of this master mixture was then incubated in separate tubes with 5µl of the lysate's sample and 2 ul of the capture code set. This hybridization mixture was incubated at 65°C for 18 hours. The hybridization mixture bind to Cartridge with a NanoString Prepstation.
|
| Scan protocol |
The cartridgenCounter™ Analysis System allows then to collect image and count barcodes.
|
| Data processing |
Raw counts were normalized to geometric mean counts of the synthetic positive controls included on the codeset. nCAAM and the geNorm algorithm was used to normalize each dataset by taking the five most stable housekeeping genes across samples and using it on all datasets.
Data were analyzed by ROSALIND® (https://rosalind.bio/), with a HyperScale architecture developed by ROSALIND, Inc. (San Diego, CA). Read Distribution percentages, violin plots, identity heatmaps, and sample MDS plots were generated as part of the QC step. Normalization, fold changes and p-values were calculated using criteria provided by Nanostring. ROSALIND® follows the nCounter® Advanced Analysis protocol of dividing counts within a lane by the geometric mean of the normalizer probes from the same lane. Housekeeping probes to be used for normalization are selected based on the geNorm algorithm as implemented in the NormqPCR R library.
|
| |
|
| Submission date |
Sep 21, 2023 |
| Last update date |
Sep 25, 2023 |
| Contact name |
Nicolas Huot |
| E-mail(s) |
nicolas.huot@pasteur.fr
|
| Organization name |
Institut Pasteur
|
| Street address |
28, rue du Dr Roux
|
| City |
Paris |
| ZIP/Postal code |
75015 |
| Country |
France |
| |
|
| Platform ID |
GPL29936 |
| Series (2) |
| GSE243731 |
SARS-CoV-2 viral persistence in lung alveolar macrophages is controlled by IFN-g and NK cells [BALF_Macro_SARS_NHP] |
| GSE243734 |
SARS-CoV-2 viral persistence in lung alveolar macrophages is controlled by IFN-g and NK cells |
|
