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| Status |
Public on May 21, 2010 |
| Title |
UT |
| Sample type |
SRA |
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| Source name |
Primary bone marrow-derived macrophages (BMDM) from Fvb mice
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| Organism |
Mus musculus |
| Characteristics |
cell type: BMDM cells (7th day of differentiation)
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| Treatment protocol |
none
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| Growth protocol |
Bone marrow cells isolated from female Fvb mice were plated in 10 cm plates in 5ml of BM-medium (high glucose DMEM supplemented with 20% low endotoxin fetal bovine serum, 30% L929-conditioned medium, 1% glutamine, 1%, Pen/Strep, 0.5% Sodium Pyruvate, 0.1% β-mercaptoethanol). Cultures were fed with 2.5 ml of fresh medium every two days.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were lysed in HB buffer (10% glycerol, 60 mM KCl, 15 mM NaCl, 1.5 mM HEPES pH 7.9, 0.5 mM EDTA) containing 0.3M sucrose and 0.8% NP40. Nuclei were then pelleted through a 0.9M sucrose cushion in HB buffer and then resuspended in 100 ml of NRB (75 mM NaCl, 20 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 50% glycerol). Transcripts were extracted by Trizol (Invitrogen) followed by manufacturer's instructions. RNA is then fragmented using divalent cations under elevated temperature. Then the cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNaseH. These cDNA fragments then go through an end repair process, the addition of a single 'A' base, and then ligation of the adapters. These products are then purified and enriched with PCR to create the final cDNA library.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
none
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| Data processing |
Alignment: sequence reads were mapped to the mouse mm9 genome using TopHat (PMID: 19289445, Illumina pipeline version 1.3). All reads that map uniquely to the genome with two or fewer mismatches were kept. Lanes 1 and 2 were sequenced up to 76bp while lanes 3 and 4 up to 55bp. For this reason all the reads were trimmed to 55bp before aligning.
Transcripts discovery and quantification was performed using Cufflinks 0.8.0 (unpublished, http://cufflinks.cbcb.umd.edu/).
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| Submission date |
Feb 17, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Iros Barozzi |
| E-mail(s) |
iros.barozzi@meduniwien.ac.at
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| Organization name |
Medical University Vienna
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| Street address |
Borschkegasse 8a
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| City |
Vienna |
| ZIP/Postal code |
1090 |
| Country |
Austria |
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| Platform ID |
GPL9250 |
| Series (1) |
| GSE20370 |
A large fraction of extragenic RNA Pol II transcription sites overlap enhancers |
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| Relations |
| SRA |
SRX017737 |
| BioSample |
SAMN00009910 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM510272_UT.cufflinks_0.8.transcripts.txt.gz |
2.2 Mb |
(ftp)(http) |
TXT |
| GSM510272_UT.tophat.sam.txt.gz |
483.6 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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