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Series GSE62187 Query DataSets for GSE62187
Status Public on Nov 24, 2014
Title An optimized microRNA scaffold increases shRNA processing and target knockdown
Organisms Gallus gallus; Homo sapiens
Experiment type Non-coding RNA profiling by high throughput sequencing
Summary Two shRNAs were placed into expression vectors harboring mir30 microRNA scaffold and an optimized scaffold where the artificial restriction sights in mir30 have been removed. After infection and selection shRNA processing was assessed by small-RNA cloning. For both shRNAs, placement into the optimized scaffold resulted in a ~two-fold increase in processing (based on smallRNA levels).
Overall design Purpose: Others have reported that the EcoRI site that was introduced to the mir30 scaffold results in decreased smallRNA processing and hence reduced target knockdown. We've developed an alternative scaffold (termed ultramir) where this site is removed. smallRNA cloning was used to determine if the movement of this sight resulted in an increase in shRNA processing.
Method: Two shRNAs (one targeting Renilla Luciferase and one targeting Human RPA3) were cloned into the original mir30 cassette the ultramir cassette. Each of the 4 constructs were infected in duplicate at single copy into cells and the cells seltected unitil infection percentages reached >90% (the shRenilla hairpin was infected into HEK293T cells and the shRPA3 construts into the Gallus gallus cell line ERC. After selection smallRNA cloning was perfromed and the amount of smallRNAs corrresponding to the two shRNAs compared to the endogenous microRNA populatlon.
Results: smallRNA levels of the two shRNAs doubled relative to the microRNA population when they were placed into the ultramir scaffold.
Contributor(s) Knott SR
Citation(s) 25435137
Submission date Oct 08, 2014
Last update date May 15, 2019
Contact name Simon Robert Vincent Knott
Organization name Cold Spring Harbor Laboratory
Department Biology
Lab Hannon
Street address 1 Bungtown Rd
City Cold Spring Harbor
State/province NY
ZIP/Postal code 11724
Country USA
Platforms (2)
GPL15520 Illumina MiSeq (Homo sapiens)
GPL19064 Illumina MiSeq (Gallus gallus)
Samples (8)
GSM1521518 shRPA3 in mir30 Replicate A
GSM1521519 shRPA3 in mir30 Replicate B
GSM1521520 shRPA3 in ultramiR Replicate A
This SubSeries is part of SuperSeries:
GSE62189 A computational algorithm to predict shRNA potency
BioProject PRJNA263394
SRA SRP048755

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE62187_Renilla_Variance_Stabalized.txt.gz 8.9 Kb (ftp)(http) TXT
GSE62187_Rpa3_Variance_Stabalized.txt.gz 4.2 Kb (ftp)(http) TXT
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Raw data are available in SRA
Processed data are available on Series record

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