|
| Status |
Public on Feb 14, 2014 |
| Title |
miR-17-5p |
| Sample type |
SRA |
| |
|
| Source name |
HEK293T transfected with miR-17-5p duplex
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
duplex: miR-17-5p
|
| Treatment protocol |
200 pmoles of synthetic biotin-labelled miR-17-5p duplex were transfected into 4 x 10^6 HEK293T cells using HiPerFect Transfection Reagent (Qiagen). Cells were incubated for 24 hours.
|
| Growth protocol |
Cells were grown in DMEM and 5% CO2 at 37oC; passaged routinely at 80% confluencey by splitting 1 in 5.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were lysed in hypotonic lysis buffer (10mM KCl, 1.5mM MgCl2,10mM Tris-Cl pH 7.5, 5mM DTT, 0.5% NP-40,60U/ML SUPERase•In (Ambion) and 1X Complete Mini protease inhibitor (Roche)). Cell debris was cleared by centrifugation (≥10,000g at 4º C for 2 minutes). The supernatant was transferred to a clean tube, and NaCl was added to a final concentration of 1M. 25μL of myOne C1 Dynabeads (Invitrogen) were pre-blocked with 1μg/μL BSA and 1μg/uL yeast tRNA (Invitrogen), and incubated with the supernatant for 30 minutes at room temperature. Beads were then washed with hypotonic lysis buffer and 1M NaCl before RNA extraction using an RNeasy Kit (Qiagen) according to manufacturer's instructions.
Total RNA was depleted of rRNA (Ribominus Kit, Invitrogen) to select mRNA. The enriched mRNA was fragmented by digestion with RNaseIII (Ambion), and purified on a Microcon YM30 column (Microcon). Fragmented mRNA was used to generate libraries as specified in the Whole Transcriptome Analysis Kit (Ambion) protocol for mRNA.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
AB SOLiD System 3.0 |
| |
|
| Data processing |
Basecalling performed with SOLiD Analysis Tools (SAT) v3.5.
Reads were mapped to the human genome (GRCh37) using the X-MATE pipeline with the parameters: max_multimatch=2; recursive_maps =50.5.0,45.5.0,40.5.0,35.3.0,30.3.0,25.1.1; run_rescue=false; map_ISAS=false;
Genome_build: GRCh37 (hg19)
Supplementary_files_format_and_content: Positive and negative strand bedGraph files were generated as a part of the X-MATE output; Scores represent the number of times a nucleotide was seen in the aligned data.
|
| |
|
| Submission date |
Feb 14, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Nicole Cloonan |
| E-mail(s) |
nicole.cloonan@qimrberghofer.edu.au
|
| Organization name |
QIMR Berghofer Medical Research Institute
|
| Lab |
Genomic Biology
|
| Street address |
300 Herston Road
|
| City |
Herston |
| State/province |
Queensland |
| ZIP/Postal code |
4006 |
| Country |
Australia |
| |
|
| Platform ID |
GPL9442 |
| Series (2) |
| GSE55058 |
Imperfect centered miRNA binding sites are common and can mediate functional repression of target mRNAs [sequencing] |
| GSE55059 |
Imperfect centered miRNA binding sites are common and can mediate functional repression of target mRNAs |
|
| Relations |
| SRA |
SRX471825 |
| BioSample |
SAMN02643352 |
