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| Status |
Public on Nov 13, 2013 |
| Title |
bonemarrow-monocyte-S3-1114542 [RNA-seq] |
| Sample type |
SRA |
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| Source name |
bonemarrow-monocyte
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| Organism |
Mus musculus |
| Characteristics |
tissue: bone marrow
cell type: monocyte
strain: C57BL/6
age: 6 weeks
gender: Female
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| Treatment protocol |
Tissue was dissociated, enriched by Percoll gradient, and cells were flow sorted.
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| Extracted molecule |
total RNA |
| Extraction 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.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
bone marrow monocyte biological replicate 3
S3
RNA-Seq (300-500bp size fractionation)
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| Data processing |
Basecalls performed using CASAVA version 1.7.0
Aligned with TopHat 1.3.1, Bowtie 0.12.7 and Ensembl 58 annotation (-G param)
FPKM calculated per sample with Cufflinks 1.0.3
Normalized expression calculated per sample by Alexa-Seq 1.17
Genome_build: mm9
Supplementary_files_format_and_content: Matrix_GeneExpression_v60_S1-6.txt, a tab delimited file with columns for gene ID and the normalized expression values per sample name
Supplementary_files_format_and_content: 11881_gene_fpkm.tsv, a tab delimited file with columns for gene ID and the normalized expression values per sample name
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| Submission date |
May 07, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Winnie W Pong |
| Organization name |
Washington University School of Medicine
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| Department |
Department of Neurology
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| Lab |
David H Gutmann Laboratory
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| Street address |
660 S. Euclid Avenue
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| City |
St Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE46688 |
F11R is a novel monocyte prognostic biomarker for malignant glioma [Seq] |
| GSE46690 |
F11R is a novel monocyte prognostic biomarker for malignant glioma |
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| Relations |
| BioSample |
SAMN02138464 |
| SRA |
SRX275699 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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