mRNA knockdown profiles for 7 gene-signature post transduction with TRCN#40273.

<p>(<b>A</b>) qRT-PCR results for panel of 4 cell lines including reporter cell line. (<b>B</b>) qRT-PCR results for panel of 6 melanoma cell lines. (<b>C</b>) qRT-PCR results for panel of 6 adenocarcinoma cell lines. (<b>D</b>) qRT-PCR results for panel of 5 cell lines derived from breast, kidney or retina. (<b>E</b>) Clustered heatmap to show mRNA knockdown levels of 7 genes across 27 distinct cell lines. (<b>F</b>) qRT-PCR results for 3 <i>DICER1</i>^mut and 3 <i>DICER1</i>^wt cell lines. Data in the bar graphs is expressed as average ± standard error.</p

Schematics of the walk-through study using TRCN#40273.

<p>Hairpin cleavage variants identified for the entire length of the oligonucleotide with a step size of 1</p

Walk-through experiment results for 78 ATS siRNA duplexes.

<p>Results of walk-through experiments measured at day 6 post transfection with synthetic siRNA duplexes using EGFPB reporter cell line. (<b>A</b>) Clustered heatmap to show % gain in EGFP signal conferred by 78 ATS siRNA duplexes, tested as singles. (<b>B</b>) Clustered heatmap to show % gain in EGFP signal conferred by siRNA duplexes segregated into three pools that of duplexes active as singles, inactive as singles or with all inclusive. Rep stands for replicate, AVG stands for average of the four replicates.</p

Schematics for Alternate Targeting Sequence Generator (ATSG).

<p>Hairpin inside the cell gets cleaved at its theoretical site and silences its target specifically. Inefficiencies in cleavage would lead to ATSG, generating random targeting sequencing which silence alternate targets, making it extremely difficult to comprehend the eventual phenotypic outcomes.</p

List of 27 total cell lines used towards confirmation of 7 gene-signature knockdowns.

1<p>Reporter cell line used in the screen and walk-through experiments.</p>2<p>Cell line used for TRCN#40273 validation performed by Sigma-Aldrich in collaboration with the Broad Institute.</p><p>wt; wild-type, mut; mutant.</p

<i>DICER1</i> depleted functional reporter & protein expression.

<p>(<b>A</b>) qRT-PCR analysis to show expression levels of <i>DICER1</i> mRNA in mutant and wild-type cell lines. (<b>B</b>) Western blot analysis to show intracellular <i>DICER1</i> protein levels in mutant and wild-type cell lines. (<b>C</b>) Bar graph showing results of western blot analysis, quantified as <i>DICER1</i> protein expression relative to expression of β-actin (ImageJ). (<b>D</b>) qRT-PCR expression studies with miR-21 as reporter of <i>DICER1</i> activity.</p