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| Status |
Public on Mar 01, 2020 |
| Title |
sEV_C |
| Sample type |
SRA |
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| Source name |
Extracellular vessicles isolated from sEV
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| Organism |
Homo sapiens |
| Characteristics |
cell line: Simian virus 40-immortalized human osteoblast cells (SV-HFO cells)
sample type: extracellular vesicles
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| Treatment protocol |
EVs were isolated from 20 mL conditioned medium by low speed centrifugation (1500 rpm, 5 minutes; 4500 rpm, 10 minutes) followed by ultracentrifugation (20,000g, 30 minutes; 100,000g, 1 hour at 4°C) of the supernatant using the SW32Ti rotor (Beckman Coulter, Fullerton, CA, USA).
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| Growth protocol |
Simian virus 40-immortalized human osteoblast cells from fetal calvaria, SV-HFO cells (26), were seeded at a density of 1 × 10e4 cells/ cm2 and cultured in α-MEM (GIBCO, Paisley, UK) supplemented with 20 mM HEPES, pH 7.5 (Sigma, St. Louis, MO, USA), streptomycin/ penicillin, 1.8 mM CaCl2 (Sigma), 10 mM β-glycerophosphate (Sigma) and 2% FCS (GIBCO) at 37°C in a humidified atmosphere of 5% CO2 for 12 - 14 days. The culture medium was replaced every 2 - 3 days. Simian virus 40-immortalized human osteoblasts from fetal limb tissue (hFOB 1.19 cells, ATCC® CRL11372™) were seeded at a density of 1.5 × 10e4 cells/ cm2 and cultured in DMEM/F12 (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 2.5 mM L-glutamine, 0.3 mg/ mL G418, streptomycin/ penicillin and 10% FCS at 34°C in a humidified atmosphere of 5% CO2 for 24 hours. hMSC-TERT cells (27) were seeded at a density of 1.5 × 10e4 cells/ cm2 and cultured in MEM (Thermo Fisher Scientific), supplemented with streptomycin/ penicillin and 10% FCS at 37°C in a humidified atmosphere of 5% CO2 for 24 hours. All cell lines were washed with 1X PBS and refreshed with their respective serum-free culture medium 24 hours prior to EV isolation.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total EV-RNA was isolated using the TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions.
Sequencing of miRNAs was performed by Illumina MiSeq with samples prepared with the NEBNext Small RNA library preparation kit (NEB), according to the manufacturer’s instructions.
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| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina MiSeq |
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| Description |
miRNA expression of extracellular vesicles isolated from svHFO replicate C
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| Data processing |
The unprocessed fastq files were generated using the Illumina casava 1.8.3 software allowing
for one mismatch in the barcode. Subsequently alignment of the reads was performed using novoalign
with the inclusion of the adapter trimming option (standard illumina adapter) Special settings that
allow for the alignment of short reads were used. Quantification of counts was done using
feature counts against the human small RNA database. Finally differential expression analysis clustering and
principle component analysis was done in the R enviroment using the DESeq2 package.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample
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| Submission date |
Jan 14, 2020 |
| Last update date |
Mar 02, 2020 |
| Contact name |
Jeroen van de Peppel |
| E-mail(s) |
[email protected]
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| Organization name |
ErasmusMC
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| Street address |
's Gravendijkwal 230
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| City |
Rotterdam |
| ZIP/Postal code |
3015 CE |
| Country |
Netherlands |
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| Platform ID |
GPL15520 |
| Series (1) |
| GSE143613 |
Identification of extracellular vesicle miRNA cargo |
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| Relations |
| BioSample |
SAMN13841730 |
| SRA |
SRX7552734 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4263744_sEV_C-sample9.txt.gz |
4.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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