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Status |
Public on Aug 09, 2019 |
Title |
IL4.eGFP_03: M2 macrophage treated with eGFP-NPs |
Sample type |
RNA |
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Source name |
Bone Marrow Derrived Macrophages
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Organism |
Mus musculus |
Characteristics |
cell type: Bone Marrow Derrived Macrophages cytokine exposure: IL4 time of cytokine exposure: 4 days nanoparticle treatment: eGFP-NPs for 1 hour
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Treatment protocol |
At day 7 ex vivo, culture media was replaced with macrophage conditioned media (macrophage complete medium supplemented with 20 ng/mL MPLA (Sigma, cat#L6895) or 20 ng/mL IL4 (eBioscience, cat# 34-8041)). BMDMs were used between 7 - 21 days ex vivo for all experiments. For nanoparticle treatment, 1 microgram of either IRF5-NPs or eGFP-NPs were co-incubated with BMDMs for 1 hours, followed with completly removal of nanoparticles and 2X wash with culture medium.
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Growth protocol |
To prepare BMDMs, bone marrow progenitor cells were harvested from mouse bone marrow cavity of femur following established protocol [Curr Protoc Immunol. 2008 Nov;Chapter 14:Unit 14.1.]. These cells were then seeded in 100 mm culture dishes in complete media (DMEM supplemented with 4.5 g/L D-glucose, L-glutamine, 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL, Glutamax 50 mL/500 mL, supplemented with 20 ng/mL M-CSF (Peprotech, cat#315-02) at a seeding density of 0.5 – 1.0 e6/ml. Cells were allowed to grow ex vivo under 37 °C, 5% CO2 for 7 days to fully derive into BMDMs.
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Extracted molecule |
total RNA |
Extraction protocol |
RNA was extracted using RNeasy Plus Universal Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Sample RNA was quantified using a NanoDrop Microvolume Spectrophotometer (Thermo Fisher, Waltham, MA) and quality checked and RINe scores determined by TapeStation (Agilent, Santa Clara, CA).
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Label |
N/A
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Label protocol |
Ligation master mix (provided by NanoString) was added to the mRNA solution and incubated under 48 °C for 5min to allow ligation, followed with purification to remove unligated molecules.
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Hybridization protocol |
The ligated RNA were co-incubated with miRNA capture probeset, miRNA reporter codeset in hybridization buffer following the protocol provided by NanoString. The hybridation happened in 65 °C for at least 12 hours.
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Scan protocol |
All samples were read on nCounter Analysis System (NanoString Technologies).
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Data processing |
Raw data was processed and checked for quality using the R/Bioconductor package NanoStringQCPro (Nickles D, Sandmann T, Ziman R and Bourgon R (2017). NanoStringQCPro: Quality metrics and data processing methods for NanoString mRNA gene expression data. R package version 1.10.0.). Expression values were normalized to the geometric mean of housekeeping genes and log2-transformed using nSolver 4.0 software (NanoString Technologies). FDR for ratio data was calculated from the p-values returned by the t-test using the Benjamini-Yekutieli method.
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Submission date |
Sep 20, 2018 |
Last update date |
Aug 09, 2019 |
Contact name |
Matthias Stephan |
Organization name |
Fred Hutchinson Cancer Research Institute
|
Street address |
1100 Fairview Ave. N., Thomas Research Buildin
|
City |
Seattle |
State/province |
WA |
ZIP/Postal code |
98109-9024 |
Country |
USA |
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Platform ID |
GPL25266 |
Series (1) |
GSE120254 |
Identify signature genes associated with nanoparticle carrying IRF5 mRNA treatment in bone marrow derrived macrophages |
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