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| Status |
Public on Feb 14, 2018 |
| Title |
secondary structure based on icSHAPE of pre-let-7 and hDicer-TRBP bound pre-let-7 |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
we resolved precusor let7 (pre-let-7) and human Dicer-TRBP complex bound precusor let7 (hDicer-TRBP-pre-let-7) secondary structure based on in vivo click selective 2'-hydroxyl acylation and profiling experiment (icSHAPE). We found the free pre-let-7 RNA is more flexible comparing with hDicer-TRBP-pre-7 in the double strand region [10-20nt and 50- 60 nt].
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| Overall design |
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. then the RNA are purified and building library as the icSHAPE protocol.
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| Contributor(s) |
Sun L, Zhang QC |
| Citation(s) |
29706542 |
| Submission date |
Feb 13, 2018 |
| Last update date |
Mar 20, 2019 |
| Contact name |
Lei Sun |
| E-mail(s) |
sunlei0227@sdu.edu.cn
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| Phone |
13121145408
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| Organization name |
Shandong University
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| Lab |
Sun Lab
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| Street address |
Shandong street
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| City |
Qingdao |
| State/province |
Shandong |
| ZIP/Postal code |
266071 |
| Country |
China |
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| Platforms (1) |
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| Samples (4)
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| Relations |
| BioProject |
PRJNA433923 |
| SRA |
SRP132769 |
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