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Status |
Public on May 20, 2024 |
Title |
MC_PGE2_2 |
Sample type |
SRA |
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Source name |
Bone Marrow
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Organism |
Mus musculus |
Characteristics |
tissue: Bone Marrow cell type: Mast cell strain: C57BL/6 treatment: 1uM PGE2 time point: 24 hours
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Treatment protocol |
Murine BMMCs (50,000 cells/well) were stimulated with 10 ng/mL of IL-33 and/or 1 µM of PGE2 for 24 hours in biologic quadruplicate. CBMCs were rested at 50,000 cells/100 uL in media containing SCF (100 ng/mL) overnight prior to stimulation with 1 µM PGE2 for 72 hours in technical duplicate
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Growth protocol |
MCs were cultured in RPMI 1640 supplemented with 10% FBS, 1% pen-strep, and 55 micromolar of 2-mercaptoethanol with 50 ng/mL SCF and 30 ng/mL IL-3 (mouse) or 10 ng/mL IL-10, 50 ng/mL IL-6, and 100 ng/mL SCF (human).
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Extracted molecule |
total RNA |
Extraction protocol |
Cells were spun down and resuspended in TCL buffer (Qiagen) with 1% 2-mercaptoethanol. Lysate containing 1,000 cells was transferred into low-bind plates for sequencing through the Broad Institute Genomics Platform SmartSeq2 libraries were prepared according to the SmartSeq2 protocol (Picelli et al, 2013, 2014) with some modifications (Trombetta et al, 2014). Briefly, total RNA was purified using RNA-SPRI beads. Poly(A)+ mRNA was converted to cDNA which was then amplified. cDNA was subject to transposon-based fragmentation that used dual-indexing to barcode each fragment of each converted transcript with a combination of barcodes specific to each sample. Barcoded cDNA fragments were then pooled prior to sequencing. Sequencing was carried out using an Illumina NextSeq500 as paired- end 2x25bp with an additional 8 cycles for each index.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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Description |
MC_PGE2_2
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Data processing |
Reads were pseudoaligned to the GrCH38 (human) or GRCm39 (mouse) assembly using Kallisto (Bray et al, 2016) with default settings. Transcript-level (human) or gene-level (mouse) expression data was imported into R using TXImport (Soneson et al, 2015) Assembly: GrCH38 (human) or GRCm39 (mouse) Supplementary files format and content: TXImport file containing abundance, counts, and length
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Submission date |
May 03, 2024 |
Last update date |
May 20, 2024 |
Contact name |
Daniel Dwyer |
E-mail(s) |
dfdwyer@bwh.harvard.edu
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Organization name |
Brigham and Women's Hospital
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Street address |
60 Fenwood Road
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City |
Boston |
State/province |
MA |
ZIP/Postal code |
02115 |
Country |
USA |
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Platform ID |
GPL24247 |
Series (1) |
GSE266501 |
Mast Cells Control Lung Type 2 Inflammation via Prostaglandin E2-Driven Soluble ST2. |
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Relations |
BioSample |
SAMN41200628 |
SRA |
SRX24450570 |
Supplementary data files not provided |
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Raw data are available in SRA |
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