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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 31, 2024 |
| Title |
Δ/DN Rep3 - US |
| Sample type |
SRA |
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| Source name |
Bone marrow derived cells
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| Organism |
Mus musculus |
| Characteristics |
tissue: Bone marrow derived cells
cell type: Bone marrow defived dendritic cells (BMDCs)
genotype: {delta}/DN
treatment: US
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| Treatment protocol |
After 8-9 days, BMDCs were harvested and seeded at 106 cells/mL for stimulation with lipopolysaccharide (LPS, E. coli O111:B4, Millipore-Sigma), polyinosinic–polycytidylic acid (poly(I:C), Millipore-Sigma), or (1→3)-β-D-glucan from Alcaligenes faecalis (Millipore-Sigma).
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| Growth protocol |
For BMDC derivation, mouse bone marrow was resuspended at 107 cells/mL and seeded dropwise at 75 μLs per well into 6-well non-tissue culture treated plates (Fisher Scientific) containing 3 mL/well of RPMI-1640 media, with 10% fetal calf serum (FCS) (both from ThermoFisher Scientific), 2 mM L-glutamine (Wisent), 100 U/mL penicillin/streptomycin (Wisent), 50 μM β-mercaptoethanol (Millipore-Sigma), and 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF, 315-03, Peprotech) The cultures were fed with fresh media at days 3 and 6.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the EZ-10 DNAaway RNA kit (Bio Basic), according to the manufacturer’s protocol, and quantified with NanoDrop (ThermoFisher Scientific). cDNA was prepared using the Moloney murine leukemia virus (MMLV) reverse-transcription kit and quantitative PCRs run on a StepOnePlus instrument with PowerSYBR master mix (all from ThermoFisher Scientific). Primers were purchased from Integrated DNA Technologies.
RNA yields and quality were assessed using Bioanalyzer (Agilent). rRNA depletion and library preparation were performed using the KAPA RNA HyperPrep Kit with RiboErase (Roche).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Tmx3_A2_US
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| Data processing |
The quality of the sequencing reads was confirmed using the FastQC tool (Babraham Bioinformatics), and low-quality bases were trimmed from the read extremities using Trimmomatic v.0.33.The reads were then mapped to the mouse UCSC mm39 reference genome assembly using Hisat2 v2.2.1. Gene expression was quantified by counting the number of uniquely mapped reads with featureCounts using default parameters.
We retained genes that had an expression level of at least 5 counts per 106 reads (CPM) in at least 3 of the samples, and performed quantile normalization with the preprocessCore package to remove batch effects. TMM normalization and differential gene expression analyses were conducted using the edgeR Bioconductor package. Dimension reduction analysis was performed using the Principal Component and Partial Least Squares regression method.
Pairwise comparisons were performed between the genotypes or between the treatments, and genes with changes in expression ≥ |1.5| fold and Benjamini-Hochberg adjusted p values ≤ 0.01 were considered significant.
For data visualization in Integrative Genomics Viewer (IGV) bigwig files were generated using a succession of genomeCoverageBed and wigToBigWig tools and scaled per 10x106 exon-mapped reads. Gene ontology (GO) enrichment analyses on differentially expressed gene clusters were performed with DAVID Bioinformatics Resources 6.8, and Gene Set Enrichment Analysis (GSEA) was performed with GSEA tool v4.3.2 using MSigDB v2022.1 with default configuration and permutation within gene sets.
Assembly: GRCm39/mm39
Supplementary files format and content: tab-delimited text files include feature counts for each sample, where rRNA have been excluded.
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| Submission date |
Jul 05, 2023 |
| Last update date |
Jan 31, 2024 |
| Contact name |
Anastasia Nijnik |
| E-mail(s) |
[email protected]
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| Phone |
514-398-5567
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| Organization name |
McGill University
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| Department |
Physiology
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| Lab |
Nijnik Lab
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| Street address |
Rm 368 - 3649 Promenade Sir William Osler
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| City |
Montreal |
| State/province |
Quebec |
| ZIP/Postal code |
H3G 0B1 |
| Country |
Canada |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE236534 |
Chromatin Binding Deubiquitinase MYSM1 in Dendritic Cell Development and Activation |
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| Relations |
| BioSample |
SAMN36306609 |
| SRA |
SRX20898059 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7549608_Tmx3_A2_US_paired_featurecounts_no_rRNA.txt.gz |
357.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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