Mus musculus
Protocols: Triplicates of single cell mouse bone marrow suspensions pooled from 3 male C57BL/6J mice in the steady state and 3 male mice that received daily intraperitoneal injections of 400 ng recombinant murine IL-33 for 7 days were first stained for Ly6G-FITC (BioLegend, 127606), CD90.2-FITC (BioLegend, 140204), NK1.1-FITC (eBioscience, 11-5941-85), CD45R-FITC (eBioscience, 11-0452-85) for 30 minutes on ice. Samples were immunomagnetically enriched for cells of interests using EasySep™ FITC positive selection kit II (Stemcell, 17682) as described by the manufacturer's instructions. The negative fractions were subsequently stained for CD29-pacblue (BioLegend, 102224), F4/80-BV510 (BioLegend, 123125), Siglec-F-BV711(BD Biosciences,740764), CD55-biotin-streptavidin-BV786(R&D, BAF5376, BD Biosciences, 563858), CD117-BB700 (BD Biosciences, 566414), Pir-A/B-PE (BioLegend, 144104) and CD200R3-PE-cy7 (BioLegend, 142212) in 1X BD Horizon™ Brilliant stain buffer (BD Biosciences, 563794) for 30 minutes on ice. Cell suspensions were washed twice and resuspended in PBS supplemented with 5nM BD Via-Probe™ Green (BD Biosciences, 565802). Mouse eosinophil stages were sorted directly into TRIzol (ThermoFisher, 15596026) using a BD FACSAria III (BD Biosciences) cell sorter with a 100μm nozzle. Sort purity was at 95 per cent or higher and samples were stored at -80°C for downstream RNA applications. For every mL of TRIzol, 200uL of chloroform was added, and the samples were vigorously mixed and incubated for 2 minutes at room temperature. Samples were centrifuged at 10,000xg for 15 minutes at 4°C to separate the phases. The RNA-containing upper aqueous phase was transferred to a new microcentrifuge tube containing 475uL of isopropanol and 2uL of glycoblue (ThermoFisher, AM9515). Samples were centrifuged at 10,000xg for 15 minutes and supernatant was discarded. One volume of 75% ethanol was added and samples were centrifuged at 10,000xg for 1 minute to precipitate the RNA and discard the supernatant. RNA pellet was resuspended in 40uL of DNase/RNase-free water for a 15-minute DNase treatment (Zymo Research, E1010). DNase treatment was followed by column-based RNA purification with the RNA Clean & concentrator-5 kit (Zymo Research, R1016). Briefly, 100uL of RNA binding buffer was added to every 50L sample and mixed thoroughly. One volume of 100% ethanol was added and the sample was transferred into a Zymo-Spin IC column in a collection tube. Columns were centrifuged at 10,000xg for 30 seconds and flow-through was discarded. The column was washed once with RNA prep buffer and twice with RNA wash buffer, following the manufacturer's instructions. RNA was eluted in 10uL of DNase/RNase-free water. Purified RNA integrity and quantity was assessed using the RNA 6000 Pico kit (Agilent) for the presence of 18s and 28s rRNA peaks. Full length cDNA was prepared from isolated RNA using SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio, 634889) following the manufacturer's instructions, with 17 cycles of cDNA amplification. Final cDNA quality was assessed using Agilent High Sensitivity DNA kit (Agilent, 5067-4626). cDNA libraries were prepared for sequencing using Nextera XT DNA library preparation kit (Illumina, FC-131-1024) using the manufacturer's instructions.
phenotype
eosinophil maturation stage IV
strain
C57BL/6J
disease
Eosinophilia
individual
6
organism
Mus musculus
organism
Mus musculus
sex
male
scientific_name
Mus musculus
common name
house mouse
organism part
bone marrow
developmental stage
adult
genotype
wild type genotype
isolate
not applicable
age
8 to 16
ENA-LAST-UPDATE
2023-04-14