|
| Status |
Public on Feb 14, 2018 |
| Title |
hDicer-TRBP-pre-let-7-DMSO |
| Sample type |
SRA |
| |
|
| Source name |
hDicer-TRBP-pre-let-7-DMSO
|
| Organism |
Homo sapiens |
| Characteristics |
group: hDicer-TRBP-pre-let-7 complex
|
| Treatment protocol |
The pre-let-7 RNA (5ʹ-UGAGGUAGUAGGUUGUAUAGUUUUAGGGUCACACCCACCACUGGGAGAUAACUAUACAAUCUACUGUCUUACC-3ʹ) was synthesized by Genescript Inc. In order to form the hairpin pre-let-7, we heated the RNA at 90 °C for 3 min then snap-cooled it on ice for 5 min. In order to reconstitute the hDicer-TRBP-pre-let-7 complex, we mixed ~200 µg (0.8 nmol) hDicer-TRBP complex and ~54 µg (2.3 nmol) pre-let-7 RNA in 50 µl of gel filtration buffer containing 2 mM Ca2+ and incubated the mixture on ice for 40 min. After this, the mixture was applied to a Superose™ 6 Increase 5/150 GL (GE Healthcare) column installed on an ÄKTAmicro (GE Healthcare) equilibrated in gel filtration buffer containing 2 mM Ca2+. Fractions containing the hDicer-TRBP-pre-let-7 complex were collected at a final concentration of ~0.4 mg/ml. NAI-N3 modified the free pre-let-7 and hDicer-TRBP-pre-let-7 complex for 10 min in 37°C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
35 µl of buffer RLT was added to stop the reaction on ice. The RNA was purified with an RNeasy mini column and eluted with 50 µl of pure water twice (100-µl final volume).
The modified RNA was captured with DIBO-biotin at 37 °C for 2h incubation and then purified with the Zymo RNA kit. It was then subjected to end repair, linker ligation and reverse transcription. Transcribed cDNA was enriched by biotin together with the RNA moiety. cDNA libraries were amplified using qPCR with about 13-15 cycles.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
| |
|
| Description |
hDicer-TRBP-pre-let-7.icshape.out
|
| Data processing |
Library strategy: icSHAPE
remove duplicete reads as icshape pipeline
trimmed reads adaptor as icshape pipeline
mapping reads to pre-let-7 with botie2 as default parameters
calculate rpkm as icshape pipeline
calulate reverse transcript stop sites as icshape pipeline
calculate icSHAPE score as icshape pipeline
Genome_build: pre-let-7
Supplementary_files_format_and_content: tab-delimited text icshape score for icshape file
|
| |
|
| Submission date |
Feb 13, 2018 |
| Last update date |
Feb 14, 2018 |
| Contact name |
Lei Sun |
| E-mail(s) |
sunlei0227@sdu.edu.cn
|
| Phone |
13121145408
|
| Organization name |
Shandong University
|
| Lab |
Sun Lab
|
| Street address |
Shandong street
|
| City |
Qingdao |
| State/province |
Shandong |
| ZIP/Postal code |
266071 |
| Country |
China |
| |
|
| Platform ID |
GPL20795 |
| Series (1) |
| GSE110516 |
secondary structure based on icSHAPE of pre-let-7 and hDicer-TRBP bound pre-let-7 |
|
| Relations |
| BioSample |
SAMN08523344 |
| SRA |
SRX3687949 |
