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Synthetic construct

ABSTRACT: Sub-genomewide shRNAs constructed using an optimized selection algorithm and microRNA backbone provide stronger evidence for follow-up studies

PROVIDER: PRJNA263392 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR1608450.fastq.gz	Fastqsanger.gz
	SRR1608460.fastq.gz	Fastqsanger.gz
	SRR1608451.fastq.gz	Fastqsanger.gz
	SRR1608461.fastq.gz	Fastqsanger.gz
	SRR1608452.fastq.gz	Fastqsanger.gz

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Sub-genomewide shRNAs constructed using an optimized selection algorithm and microRNA backbone provide stronger evidence for follow-up studies

Project description:Sub-genomewide shRNA libraries were constructed using the current RNAi consortium constructs as well as using the DSIR (siRNA algoirthm) and a novel shRNA specific algorithm (shERWOOD). All libraries were placed into mir30 expression vectors. The shERWOOD libraries were also placed in a vector harboring an optimized mir cassette (ultramir). Each library was screened using the pancreatic cell line A385. A concensus set of essential genes identified as the set for which two shRNAs depleted in each of the libries. For these genes, a great percentage of shERWOOD seletected shRNA depleted. In addition the placement of shERWOOD selected constructs into ultramir scaffoled increased the rate of shRNA depletion for essential genes further. Purpose: shRNA screens were carried out using various library construction strategies to identify the strategy that provides the best shRNA screening results. Method: Libraries were constructed using the TRC shRNA set as well as shRNAs identified using the DSIR and shERWOOD algorithms. shRNA libraries were cloned into mir30 expression vectors. shERWOOD shRNAs were also cloned into an expression vector harboring an optimized microRNA scaffold termed ultramir. Each resultant library was screened using the pancreatic cell line A385. Each library was analyzed separately to identify a set of genes where at least two shRNAs depleted. These gene sets were intersected to develop a set of essential genes. Results: The shERWOOD shRNA libraries provided the highest number depleting shRNAs for each essential gene. Further these shRNAs depleted to a greater extent than did the shRNAs from the other libraries. When shERWOOD libraries were placed into the ultramir cassette a greater number of shRNAs per essential gene depleted.

2014-11-24 | E-GEOD-62185 | biostudies-arrayexpress

Sub-genomewide shRNAs constructed using an optimized selection algorithm and microRNA backbone provide stronger evidence for follow-up studies

2014-11-24 | GSE62185 | GEO

synthetic construct

Project description:shRNA Sensor Analysis of DSIR Selected shRNAs

| PRJNA263390 | ENA

shRNA Sensor Analysis of 5' U converted shRNAs reveals 3' target bias exists in addition to 5' guide bias

2014-11-24 | GSE62186 | GEO

Influenza B virus

Project description:Development of an optimized influenza B virus vaccine backbone

| PRJNA470645 | ENA

An optimized microRNA scaffold increases shRNA processing and target knockdown

Project description: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).

2014-11-24 | GSE62187 | GEO

shRNA off target analysis via RNAseq

Project description:shRNAs were assessed for off-target effects by comparing the gene expression profiles of cells that they had been infected into. shRNAs designed with the shERWOOD algorithm and house in the ultramir microRNA scafold were found to have very little off targeting.

2014-11-24 | GSE62184 | GEO

shRNA Sensor Analysis of 5' U converted shRNAs reveals 3' target bias exists in addition to 5' guide bias

Project description:shRNAs selected with the shERWOOD algorithm were converted to have a U at the 5' end of their guide. When endogenous 1U shRNAs were compared to artificial shRNA via the sensor algorithm, the endogenous shRNAs were found to be more efficacious. Purpose: Structural studies have hinted that the 5' end of shRNA guides is engulfed in the RISC complex. It has also been reported that shRNAs with a 5' U are more efficacious than those with other 5' caps. We wished to determine whether replacement of shRNA guide 5' nucleotides with a U, regardless of the corresponding target base, would increase their efficacy. Method: For each gene in the "druggable genome" 10 shRNAs were selected with the shERWOOD algorithm. In each case the score was assessed as if the guide had a 5' U. Sensor constructs were designed pairing 1U-guide shRNAs with their endogenous target. shRNAs were assessed for efficacy via the shRNA sensor assay (Fellmann et al. Mol Cell 2011). Results: shRNAs with artificial 5' Us were found to be less efficacious than those with an endogenous 5' U,

2014-11-24 | E-GEOD-62186 | biostudies-arrayexpress

synthetic construct

Project description:shRNA Sensor Analysis of 5' U converted shRNAs reveals 3' target bias exists in addition to 5' guide bias

| PRJNA263391 | ENA

synthetic construct

Project description:An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments

| PRJNA268435 | ENA