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| Status |
Public on May 21, 2024 |
| Title |
Stage_II_Steady_State_2 |
| Sample type |
SRA |
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| Source name |
Bone Marrow
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Bone Marrow
cell type: Bone marrow eosinophil developmental stages
treatment: steady state
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| Treatment protocol |
Single cell suspensions of human bone marrow 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. Cell suspensions were washed twice and resuspended in PBS supplemented with 5nM BD Via-Probe™ Green (BD Biosciences, 565802). Four maturation stages of eosinophils 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.
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| Extracted molecule |
total RNA |
| Extraction protocol |
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 50µL 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.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Sequenced reads were aligned and mapped to the human genome (UCSC hg19) with RNA-Seq Alignment (Version:2.0.2) using STAR_2.6.1a on BaseSpace (https://basespace.illumina.com).
Normalization and analysis of the obtained data was performed using DESeq2 R package (https://bioconductor.org/packages/release/bioc/html/DESeq2.html)
Assembly: human genome (UCSC hg19)
Supplementary files format and content: Tab-delimited file describing the number of reads mapped to each gene as obtained with RNA-Seq Alignment (Version:2.0.2) on BaseSpace (https://basespace.illumina.com).
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| Submission date |
Apr 16, 2023 |
| Last update date |
May 21, 2024 |
| Contact name |
Christophe J Desmet |
| E-mail(s) |
christophe.desmet@uliege.be
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| Organization name |
University of Liege
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| Lab |
Cellular and Molecular Immunology
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| Street address |
B34, Avenue de l'Hopital 1
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| City |
Liege |
| ZIP/Postal code |
4000 |
| Country |
Belgium |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE229822 |
RNA-seq of the 4 stages of maturation of eosinophils in healthy human bone marrow. |
| GSE249011 |
Bulk and single-cell RNA sequencing of human and murine eosinophil lineage cells |
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| Relations |
| BioSample |
SAMN34208045 |
| SRA |
SRX19982708 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7178563_6NGS22Y301.counts.genes.txt.gz |
129.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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