|
| Status |
Public on Jan 31, 2015 |
| Title |
sw2 |
| Sample type |
SRA |
| |
|
| Source name |
malignant chondrocyte
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SW1353
tissue type: Human bone chondrosarcoma, fibroblast-like cell line
molecule subtype: small RNA
|
| Treatment protocol |
no special treatment
|
| Growth protocol |
SW1353 chondrosarcoma cells were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies) containing 10% (volume/volume) fetal bovine serum (Sigma Aldrich), 2 mM glutamine and 1% (volume/volume) penicillin-streptomycin. Cells were fed every other day and maintained at 37oC in an atmosphere of 5% CO2. After 21 days of culture, cells were collected for total RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the miRCURY™ RNA Isolation Kit - Tissue (Exiqon).
Total RNA (1µg) was ligated to adenylated 3’ HD adapter by using T4 RNA ligase 2 (truncated T4 RNA ligase). The ligated product was cleaned and concentrated by Zymo RNA cleaning and concentrating kit. The remaining adapter was de-adenylated by adenylase and degraded by exonuclease. Then the 3’ ligated products were ligated to 5’ HD adapter by using T4 RNA ligase 1, and the reaction product was further cleaned and concentrated by the Zymo RNA cleaning and concentrating kit. The concentrated RNA being ligated to both 5’ and 3’ HD adapters were reverse transcribed into cDNA and the cDNA was amplified by using Phusion DNA polymerase. The amplified products were run on 6-8% of PAGE gel. The band around the size of 147bp was cut and the PCR products, the library, were extracted from the gel and were sent for sequencing.
|
| |
|
| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
As an initial quality filter, all reads containing Ns were discarded.
The adapter removal step was conducted using the UEA sRNA workbench [Stocks et al, 2012]. The 3' adapter sequence was identified using the first 6 nt. The HD component of the adapter (first 4 and last 4 nucleotides) was subsequently removed.
The reads were mapped full length, no mis-matches or gaps allowed to the reference H. sapiens genome using PatMan [Prueffer et al, 2008]
The miRNAs were identified using the miRCat and miRprof tool within the UEA workbench.
The computational prediction of the targets was conducted using the plant target prediction tool within the UEA sRNA Workbench.
Genome_build: Homo sapiens genome, ver 38, GRCh38
Supplementary_files_format_and_content: csv file containing the sRNA expression levels.
|
| |
|
| Submission date |
Jul 11, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Irina Mohorianu |
| E-mail(s) |
irina.mohorianu@paediatrics.ox.ac.uk
|
| Organization name |
University of Oxford
|
| Department |
Medical Sciences Division
|
| Lab |
Oxford Vaccine Group
|
| Street address |
Headington
|
| City |
Oxford |
| ZIP/Postal code |
OX3 7LE |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE59331 |
An improved protocol for small RNA library construction by using High-Definition adapters [cell line] |
|
| Relations |
| BioSample |
SAMN02910066 |
| SRA |
SRX651007 |
