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SRX275699: GSM1134054: bonemarrow-monocyte-S3-1114542 [RNA-seq]; Mus musculus; RNA-Seq
1 ILLUMINA (Illumina HiSeq 2000) run: 85M spots, 17G bases, 10.3Gb downloads

Submitted by: Gene Expression Omnibus (GEO)
Study: F11R is a novel monocyte prognostic biomarker for malignant glioma [Seq]
show Abstracthide Abstract
Objective: Brain tumors (gliomas) contain large populations of infiltrating macrophages and recruited microglia, which in experimental murine glioma models promote tumor formation and progression. Among the barriers to understanding the contributions of these stromal elements to high-grade glioma (glioblastoma; GBM) biology is the relative paucity of tools to characterize infiltrating macrophages and resident microglia. In this study, we leveraged multiple RNA analysis platforms to identify new monocyte markers relevant to GBM patient outcome. Methods: High-confidence lists of mouse resident microglia- and bone marrow-derived macrophage-specific transcripts were generated using converging RNA-seq and microarray technologies and validated using qRT-PCR and flow cytometry. Expression of select cell surface markers was analyzed in brain-infiltrating macrophages and resident microglia in an induced GBM mouse model, while allogeneic bone marrow transplantation was performed to trace the origins of infiltrating and resident macrophages. Glioma tissue microarrays were examined by immunohistochemistry, and the Gene Expression Omnibus (GEO) database was queried to determine the prognostic value of identified microglia biomarkers in human GBM. Results: We generated a unique catalog of differentially-expressed bone marrow-derived monocyte and resident microglia transcripts, and demonstrated that brain-infiltrating macrophages acquire F11R expression in GBM and following bone-marrow transplantation. Moreover, mononuclear cell F11R expression positively correlates with human high-grade glioma and additionally serves as a biomarker for GBM patient survival, regardless of GBM molecular subtype. Significance: These studies establish F11R as a novel monocyte prognostic marker for GBM critical for defining a subpopulation of stromal cells for future potential therapeutic intervention. Overall design: Total RNA was isolated from three independently-generated sets of flow sorted bone marrow monocytes (CD11b+ CD45high CD115+ Ly6G- cells) and brainstem microglia (CD11b+ CD45low CD115low Ly6G- cells) for Illumina RNA-Seq, and two additional pools were subsequently generated and submitted for Affymetrix Mouse Exon 1.0ST microarray. Two of the RNA-Seq samples were additionally analyzed by the microarray, for a total of 6 samples (3 monocyte, 3 microglia) in each platform. Data outputs were analyzed by two analysis methods each (RNA-Seq data: ALEXA-Seq and Cufflinks; microarray data: Partek and Aroma). All four lists were merged into a new high-confidence gene list of transcripts that were significantly differentially expressed (DE) in three out of the four analyses. In this dataset, we includeRNA-Seq data obtained from flow sorted mouse bone marrow monocytes and brainstem microglia.
Sample: bonemarrow-monocyte-S3-1114542 [RNA-seq]
SAMN02138464 • SRS418983 • All experiments • All runs
Organism: Mus musculus
Library:
Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Total RNA was isolated from snap-frozen sorted cell pellets using TRIzol-chloroform extraction, resuspended in 10µl Ambion Nuclease-free water (Life Technologies). For cDNA synthesis, we added 5µl of isolated RNA into the Ovation® RNA-Seq method.  500ng cDNA was fragmented using covaris shearing and processed for Illumina library construction with the Illumina paired-end LT indexing protocol as previously published (Mardis et al., 2009; Govindan et al., 2012). Each library was sequenced on the Illumina HiSeq, generating between 15-22Mbp per lane. RNA-Seq (300-500bp size fractionation): cDNA samples were constructed into Illumina libraries according to the manufacturer’s protocol (Illumina Inc, San Diego, CA) with the following modifications: 1) DNA was fragmented using Covaris S2 DNA Sonicitor using the following condtions: Duty Cycle: 5, Intensity: 4, Cycles/Burst: 200, Time: 90sec (Covaris, Inc. Woburn, MA). Fragment sizes ranged between 100 and 500bp. 2) Illumina adapter-ligated DNA was amplified in a single 50ml PCR for five cycles. 3) Solid Phase Reversible Immobilization (SPRI) bead cleanup was used to purify the PCR and select for 300-500bp fragments. We added 0.8 volumes of the AmpureXP solution (preformulated with polyethylene glycol and sodium chloride) to size select DNA molecules greater than 300 bp. The size-fractioned cDNA was washed three times with 750 µl of 70% ethanol, the beads were dried, and the cDNA library was eluted off the beads by adding 20µl 10 mM Tris-HCl (pH 8.0). The size-fractioned libraries were assayed using the Agilent BioAnalyzer High Sensitivity DNA chips.
Experiment attributes:
GEO Accession: GSM1134054
Links:
External link:
Runs: 1 run, 85M spots, 17G bases, 10.3Gb
Run# of Spots# of BasesSizePublished
SRR84728484,996,32317G10.3Gb2013-11-14

ID:
390443

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