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Figure S1. TDMD is specific and robust. (A) Schematics of the two distinct lentiviral 4x target transgenes used in (B). (B) TDMD of miR-132 (means ± s.e.m.; n = 4) calculated after transducing neurons with the 4x miR-132 targets indicated in (A). (C) Sequencing analysis of miR-132 species in total RNA from hippocampal neurons (DIV16) transduced with the indicated targets. The heatmaps depict isoform-specific log2 relative expression levels, mean normalized across all samples. The isoforms are sorted based on the first principal component calculated from the full dataset. A bar plot to the right of the heatmap indicates the absolute log2 expression levels averaged over all samples. Isoform sequences are displayed by color coding. A solid black line indicates the limit of the genomically encoded sequence. (D) Comparison of RT-qPCR (Taqman assay) a n d small RNA sequencing. RT-qPCR detects 94.5 % reduction in miR-132 levels in total RNA from neurons infected for 6 days with a WT 4x target relative to a mut target. Small RNA sequencing detects a similar reduction in mature miR-132 levels, and a slightly lower reduction in the level of all miR-132 isoforms. This indicates that RT-qPCR detects mainly the mature miR-132 isoform, which is the one mostly affected by the target at steady state levels. The sum of all other miR-132 isoforms are reduced to a lower extent by the 4X target, but, because they are overall less abundant, a similar overall TDMD is detected when quantifying all miR-132 isoforms together. Shown are means ± s.e.m.; n = 2. For deep sequencing, the two biological replicates were pooled into the same library preparation. (E) Experiment comparing RT-qPCR (Taqman assay) vs. Northern blot. Both techniques detect similar reduction in mature miR-132 levels in total RNA from neurons infected for 6 days with a WT 4x target relative to a mut target. This confirms that mature miR-132 is is the most abundant isoform, and also the one predominantly affected by that target. Shown means ± s.e.m. of one experiment with two technical replicates. The technical replicates were pooled for the northern blot analysis. 4X miRNA binding site