|
| Status |
Public on Oct 11, 2011 |
| Title |
Small RNAs from kidney tissue of transgenic mice expressing Mos inverted repeat (MosIR) |
| Sample type |
SRA |
| |
|
| Source name |
kidney tissue, MosIR
|
| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6
tissue: kidney
expression: Mos inverted repeat
|
| Treatment protocol |
Cell lines were transiently transfected with 1 microgram of pCag-EGFP_MosIR plasmid (per well in 6-well plate) using TurboFect (Fermentas) trasnfection reagent.
|
| Growth protocol |
Cell lines were grown under standard tissue culture conditions (DMEM + 10% FCS). Transgenic mice were maintained under NIH guidelines.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total small RNA was isolated using the mirPremier microRNA Isolation Kit (Sigma-Aldrich). Further sample processing and SOLid sequencing was performed by Seqomics Ltd. (Hungary). Briefly, adaptor was hybridized and ligated to each sample (10 ul 2x ligase buffer, 2 ul Adaptor mix A, 3 ul hybridization solution, 3 ul RNA sample, 2 ul ligation enzyme mix; incubate O.N. at 16°C). Adapter-ligated RNA was subjected to reverse transcription (4 ul 10x RT buffer, 2 ul dNTP mix, 1 ul ArrayScript RT, 13 ul water; incubate 30 min at 42°C ) followed by RNase H digestion (add 1 ul RNase H to 10 ul cDNA and incubate 30 min at 37°C). A 1 µL aliquot of cDNA library was used as a template for PCR using SOLiD PCR primers and AmpliTaq DNA polymerase. PCR products were run on a native polyacrylamide gel (1´ TBE, 6% 19:1 acrylimide:bisacrylamide). 105- to 150-nt products were excised, eluted, precipitaced and finally eluted into 20 µL nuclease-free water. Purified amplicons were sequenced using an ABI SOLiD 3 Plus instrument.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
AB SOLiD System 3.0 |
| |
|
| Description |
Total small RNAs from kidney tissue of transgenic mice expressing an inverted repeat from mouse Moloney sarcoma oncogene (Mos) gene (MosIR).
Transgenic mice were generated by injecting linearized DNA into the male pronuclei of a C57BL/6 x BALB/c 1-cell embryo. Resulting mice were further crossed to C57BL/6 mice for at least 4 generations before being used in our experiments.
The pCag-EGFP_MosIR plasmid contains an inverted repeat from the mouse Mos (Moloney sarcoma oncogene) gene that is inserted into the 3'-UTR of the EGFP reporter gene, which is expressed using a promoter composed of the CMV enhancer and β-actin core promoter.
|
| Data processing |
The sequencing and data processing were performed by Seqomics (Hungary). Reads of 18-30 nt length were mapped to the pCag-EGFP_MosIR plasmid allowing up to 1 mismatch.
Instrument Controller Software 3.5.0
Fluidics 1.12.4
Imager 1.9.20
Protocols 5P_v8.2.1
Scripts 5P_v8.3.0
Interlock FW 58.00
SETS 3.5r0.40.38126M
|
| |
|
| Submission date |
Jan 12, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Radek Malik |
| E-mail(s) |
malikr@img.cas.cz
|
| Organization name |
Institute of Molecular Genetics of the ASCR
|
| Department |
Dep. of Epigenetics Regulations
|
| Street address |
Videnska 1083
|
| City |
Prague 4 |
| ZIP/Postal code |
CZ-14220 |
| Country |
Czech Republic |
| |
|
| Platform ID |
GPL9318 |
| Series (1) |
| GSE26577 |
Ubiquitous expression of long dsRNA in mice causes mainly RNAi effects in the oocyte |
|
| Relations |
| SRA |
SRX038815 |
| BioSample |
SAMN00191472 |
