(a-f) Western blot validation of de novo SplashRNA predictions. All shRNAs were expressed using LEPG at single-copy conditions. β-Actin (Actb, ACTB) was used for normalization.
(a) Immunoblotting of Pbrm1 in NIH/3T3s (median KD: 97%, median SplashRNA score: 1.7).
(b) Immunoblotting of Rela in NIH/3T3s (median KD: 90%, median SplashRNA score: 1.1).
(c) Immunoblotting of Bcl2l11 in NIH/3T3s (median KD: 97%, median SplashRNA score: 0.7).
(d) Immunoblotting of Axin1 in NIH/3T3s (median KD: 95%, median SplashRNA score: 1.3).
(e) Schematic of the multiple human NF2 transcript variants. NF2 has nine variants with an intersection of only 198 nucleotides, excluding the 5’UTR, rendering the prediction task especially difficult due to limited sequence space.
(f) Predicting miR-E shRNAs for extremely short transcripts. Immunoblotting of NF2 in A375s transduced with the indicated shRNAs targeting all nine NF2 variants (median KD: 89%, median SplashRNA score: 0.6).
(g) Comparison of SplashRNA and DSIR predictions against CRISPR-Cas9 mediated suppression of Cd9 in mouse embryonic fibroblasts (MEFs). Shown are normalized (relative to the indicated controls) median anti-Cd9-APC fluorescence intensities of RRT-MEFs and CRT-MEFs expressing the indicated shRNAs or sgRNAs (Online
). The six top-scoring predictions from DSIR + Sensor rules (DSIR) or SplashRNA (ordered according to their respective scores) were compared to six sgRNA sequences (Supplementary Table 2). *, Cd9.1137 is the top prediction from both algorithms and was plotted twice for clarity. While DSIR predictions triggered Cd9 knockdown with variable efficacy, SplashRNA predictions consistently induce strong Cd9 suppression, closely approaching knockout conditions.
(h) Transfer function of SplashRNA score versus protein knockdown for all 62 de novo predicted shRNAs validated by immunofluorescence (Supplementary Table 2). Green triangles indicate the minimum knockdown for 80% of the predictions for a given SplashRNA score bin. Bins were defined to have a width of 0.5 with the leftmost bin starting at 0.25. For the bin centered on SplashRNA score = 1, 80% of predictions showed at least 86% protein knockdown. The expected knockdown for the top 80% of predictions (e.g. 4/5 shRNAs) increases with the SplashRNA score. Overall, 91% of predictions with a SplashRNA score >1 showed more than 85% protein knockdown.
(i) Uncropped images of Pten (Figure 2d) and Bap1 (Figure 2e) western blots, and their respective β-Actin controls. Pten predicted molecular weight (MW): 47 kDa; MW validated by Cell Signaling Technology: 54 kDa. Bap1 predicted MW: 80 kDa; MW validated by Bethyl Laboratories: 80-95 kDa. β-Actin MW validated by Sigma-Aldrich: 42 kDa.