|
| Status |
Public on Jul 11, 2017 |
| Title |
H413_2 |
| Sample type |
SRA |
| |
|
| Source name |
HNSCC cell line: small extracellular vesicles
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: H413
culture media: DMEM-F12
source: small extracellular vesicles
|
| Growth protocol |
Cells were cultivated in supplier-recommended media with 10% fetal bovine serum (FBS) that was super-depleted of exosomes via 18 hour ultracentrifugation at 100,000g (verified via nanoparticle tracking analysis) and 1% penicillin/streptomycin at 37°C with 5% CO2 in 150 cm2 flasks with 25mL media. To achieve adequate volume for exosome isolation, cells were cultured in 2-pair sets of flasks in triplicate (6 flasks total per cell-line). Culture media were replaced approximately 48 hours prior to cells reaching 80-90% confluence; media and cells were respectively collected after 48 hours. The media from each 2-flask pair were combined (50mLtotal). After media was harvested, cells were detached from each flask and pooled for each cell-line.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from each exosome pellet using the miRNeasy Micro kit (Qiagen, Valencia, CA) according to the manufacturer’s suggested protocol; total RNA was also extracted from the pooled cells for each respective cell-line the miRNeasy Micro Kit (Qiagen), with 7.5 x105 cells in each reaction.
The NEBNext small RNA sample library preparation kit (New England BioLabs, Ipswich, MA) was used with 20 ng to 1 µg of total RNA in a 6-µL solution as input, following the manufacturer’s protocol with modification of library size selection
|
| |
|
| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 1000 |
| |
|
| Description |
microRNA
|
| Data processing |
Base calling software: Casava 1.8
Preprocessing: Sequence reads were pre-processed to remove adapters and retain only 16-30 base pair- length reads
Alignment: Reads were aligned to the reference human genome (hg19) using the Bowtie aligner
Counts: The reads aligning to each known miRNA were counted using Bioconductor packages for next-generation sequencing data analysis based on miRNA definitions in miRBase database
Normalized abundance measures: RPKM
Genome_build: hg19
Supplementary_files_format_and_content: The processed data file is in .csv format and contains a table of miRNA transcripts (rows) and raw counts and normalized abundance (RPKM) data for each sample (columns)
|
| |
|
| Submission date |
Jul 12, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Scott M Langevin |
| E-mail(s) |
langevst@uc.edu
|
| Phone |
513-558-1066
|
| Organization name |
University of Cincinnati College of Medicine
|
| Department |
Department of Environmental Health
|
| Lab |
Langevin
|
| Street address |
160 Panzeca Way, ML0056
|
| City |
Cincinnati |
| State/province |
OH |
| ZIP/Postal code |
45267 |
| Country |
USA |
| |
|
| Platform ID |
GPL15433 |
| Series (1) |
| GSE84306 |
Comprehensive microRNA-sequencing of small extracellular vesicles derived from head and neck carcinoma cells reveals common secretion profiles |
|
| Relations |
| BioSample |
SAMN05381037 |
| SRA |
SRX1936150 |
