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Sample GSM8248505

Query DataSets for GSM8248505

Status

Public on May 20, 2024

Title

MC_IL33_2

Sample type

SRA

Source name

Bone Marrow

Organism

Mus musculus

Characteristics

tissue: Bone Marrow
cell type: Mast cell
strain: C57BL/6
treatment: 10 mg/mL IL-33
time point: 24 hours

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

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).

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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

MC_IL33_2

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

Submission date

May 03, 2024

Last update date

May 20, 2024

Contact name

Daniel Dwyer

E-mail(s)

dfdwyer@bwh.harvard.edu

Organization name

Brigham and Women's Hospital

Street address

60 Fenwood Road