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Status |
Public on Mar 01, 2020 |
Title |
MN2: Monocyte RFP- 2 |
Sample type |
RNA |
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Source name |
non-infected (RFP-) monocytes
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Organism |
Mus musculus |
Characteristics |
strain: C57Bl/6 Sex: Female age: 8 weeks time post-infection: 2 weeks parasite: Leishmania amazonensis tissue: skin
|
Extracted molecule |
total RNA |
Extraction protocol |
Monocytes were sorted out of the skin mice using FACSAria II and RNA was extracted using Qiagen Rneasy mini kit according to the manufacture's protocol.
|
Label |
n/a
|
Label protocol |
RNA was not labeled. 30ng/ul of RNA was converted to cDNA to follow the NanoString manufacture's protocol for the hybridization
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Hybridization protocol |
cDNA samples were hybridized to capture the reporter CodeSet and the capture ProbeSet provided by NanoString forming the target-probe complex. The reporter probes are molecular barcodes provided by NanoString. The samples were placed in a pre-heated 65°C thermal cycler and incubated for 20h to allow the hybridization.
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Scan protocol |
Scanning was conducted using the nCounter Digital Analyser acording to the manufacture's instructions. In general, it collects data by taking images of the immobilized fluorescent reportes in the sample cartridge with a CCD camera trough a microscope objective lens. The images are processed and each lane generates one RRC (Reporter Code Count) file that contains the number of reporters counted.
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Data processing |
All analysis were done in R version 3.6.0. Raw RCC files were loaded into R and quality analysis was done with NanoStringQCPro1.16.0. For principal component analysis (PCA) and visualization of gene counts the data was normalized with a variance stabilizing transform using NanoStringNorm. PCA was done with the built in R function, prcomp. Differential expression was done with NanoStringDiff 1.14.0, which uses a negative binomial model and includes an empirical Bayes shrinkage procedure to estimate dispersion (Wang et al., 2016). A 2 x 2 factorial model was used and the effect of infection was tested (p < 0.05) on samples.
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Submission date |
Feb 27, 2020 |
Last update date |
Mar 02, 2020 |
Contact name |
Nathan Peters |
E-mail(s) |
ncpeters@ucalgary.ca
|
Organization name |
University of Calgary
|
Department |
Microbiology, Immunology and Infectious Diseases
|
Street address |
3330 Hospital Dr NW
|
City |
Calgary |
State/province |
AB |
ZIP/Postal code |
T2N4N1 |
Country |
Canada |
|
|
Platform ID |
GPL20885 |
Series (1) |
GSE146022 |
Th1/Th2 cross-regulation controls early Leishmania infection in the skin by modulating the size of the permissive monocytic host cell reservoir |
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