characteristics.text: 2024-01-01
characteristics.characteristic: ENA first public
characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: SAMEA112901098
characteristics.characteristic: External Id
characteristics.text: Laboratory of Cellular and Molecular Immunology GIGA-Research Center, Liege University
characteristics.characteristic: INSDC center name
characteristics.text: 2023-04-14T14:16:57Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS14907235
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12894:Stage_I_Steady_State_2
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: human
characteristics.characteristic: common name
characteristics.text: Protocols: Human bone marrow single cell suspensions were first stained with Lineage-cocktail (BioLegend, 348801) for 30 minutes on ice. Samples were immunomagnetically enriched for cells of interests using EasySep™ FITC positive selection kit II (Stemcell, 17682) as described in the manufacturer’s instructions. The negative fractions were next stained for CD84-BV421 (BD Biosciences, 566904), FcεR1α-BV510 (BD Biosciences, 747786), CD11b-BV711 (BioLegend, 101242), CD45-BV786 (BD Biosciences, 563204), Siglec-8-BB700 (BD Biosciences, 747867), CD38-PE (BioLegend, 356604), CD200R-PE-cy7 (BioLegend, 329312), CCR3-APC (Miltenyi, 130-123-300), and CD66b-APC-cy7 (BioLegend, 305126) in 1X BD Horizon™ Brilliant stain buffer (BD Biosciences, 563794) for 30 minutes at room temperature. Human cells suspensions were washed twice and resuspended in PBS supplemented with 5nM BD Via-Probe™ Green (BD Biosciences, 565802). Human eosinophil stages were sorted directly into TRIzol (ThermoFisher, 15596026) or into FACS buffer for bright-field microscopy 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.
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: normal
characteristics.characteristic: disease
characteristics.text: 2
characteristics.characteristic: individual
characteristics.text: not applicable
characteristics.characteristic: isolate
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: bone marrow
characteristics.characteristic: organism part
characteristics.text: eosinophil maturation stage I
characteristics.characteristic: phenotype
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: Stage_I_Steady_State_2
characteristics.characteristic: title