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| Status |
Public on May 05, 2011 |
| Title |
RNA-Seq on euploid isolated EPCs |
| Sample type |
SRA |
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| Source name |
Euploid_EPC_seq
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Circulating endothelial progenitor cells
genotype: euploid
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| Treatment protocol |
Isolated cells were not treated with any drug/exonegous chemical
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| Growth protocol |
Total mononuclear cells were isolated by density gradient centrifugation of peripheral blood samples on Histopaque-1077 (Sigma). Cells were washed twice with PBS, plated on culture dishes pre-coated with gelatin and fibronectin and maintained in endothelial growth medium-2 (EGM2; Cell Systems). Cells were cultured at 37°C with 5% CO2 in a humidified atmosphere. After four days, non-adherent cells were removed and adherent cells were collected for RNA isolation.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from endothelial progenitor cells by standard Trizol protocol. Integrity and quantity of RNA was evaluated by Experion (Biorad) following the manufacturer's instructions. Ribosomal RNA depletion was performed on 10μg of total RNA by using magnetic beads (RiboMinus Eukaryote Kit for RNA-Seq, Invitrogen) according to the manufacturer's protocol. 10 μg of total RNA were incubated at 72°C for 5 min with sequence-specific 5'-biotin labeled oligonucleotide probes. Streptavidin-coated RiboMinus Magnetic Beads were used to capture rRNA-probes to discard. The efficiency of rRNA depletion was evaluated on the Experion. RNA was fragmented with RNase III and, after cleanup with RiboMinus Concentration Module (Invitrogen) according to the manufacturer's protocol, and quantified on Qubit Fluorometer (Invitrogen). The appropriate size distribution of fragmented RNA was evaluated on the Experion. 100 ng of fragmented RNA was hybridized and ligated to double stranded oligonucleotides adapter suited for the 5' SOLiD System sequencing. Reverse transcription was performed using ArrayScript Reverse Transcriptase. Purified cDNA samples were denatured on 6% TBE Urea gel, and size selection (150-250 bp) was performed. PCR amplification on gel slices was then performed using AmpliTaq DNA Polymerase, and yield of purified PCR products was assessed on the Qubit Fluorometer and NanoDrop spectrophotometer (Invitrogen). Size distribution of cDNA libraries was evaluated on the Experion.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
AB SOLiD System 3.0 |
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| Description |
rRNA-depleted total RNA
Massive-scale transcriptome analysis of cEPCs on total RNA depleted from abundant rRNA species
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| Data processing |
Total reads produced were filtered out accordingly to quality values, and secondly, to the mapping to adapters and to rRNA. RNA-MATE software version 1.1 was used to map the usable reads either to the genome (hg19) and to a custom-designed library of exon-junction sequences containing 2010842 sequences. RNA-MATE mapping to the genome was customized using the following cycles: 50.5.1, 45.4.1, 40.4.1, 35.3.1, 30.3.1, 25.1.1, 23.0.0, 22.0.0, 21.0.0 In the pipeline, all multiple reads (with at most 10 mapping positions) underwent the rescue procedure with default parameters. Bedgraph and BED files were automatically produced after the mapping.
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| Submission date |
Feb 22, 2011 |
| Last update date |
Jun 11, 2013 |
| Contact name |
Valerio Costa |
| Organization name |
Institute of Genetics and Biophysics “A. Buzzati-Traverso”, CNR, Naples, Italy
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| Lab |
Human Genetics Diseases
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| Street address |
Via P. Castellino 111
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| City |
Naples |
| ZIP/Postal code |
80131 |
| Country |
Italy |
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| Platform ID |
GPL9442 |
| Series (1) |
| GSE27443 |
RNA-Seq of rRNA depleted circulating endothelial progenitor cells (cEPCs) in trisomy 21 and euploid samples |
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| Relations |
| BioSample |
SAMN02197035 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM678310_Euploid.BED.gz |
2.0 Mb |
(ftp)(http) |
BED |
| GSM678310_Euploid_negative.bedgraph.gz |
25.6 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM678310_Euploid_negative.starts.txt.gz |
19.1 Mb |
(ftp)(http) |
TXT |
| GSM678310_Euploid_positive.bedgraph.gz |
26.3 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM678310_Euploid_positive.starts.txt.gz |
15.8 Mb |
(ftp)(http) |
TXT |
| Processed data provided as supplementary file |
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