Evidence that encoded shRNA are not induced in the absence of Doxycycline <i>in vitro.</i>

<p>(A) A lentiviral library containing 27,500 unique barcodes was used to transduce HCT-116 cells at a MOI of 0.3. After 72 hours, of puromycin selection, cells were continuously passaged for 15 days in the absence of Doxycycline. Day 0 and Day 15 cell aliquots were obtained and total DNA from both time points were subjected to the barcode high-throughput sequencing retrieval procedure. (B) Correlation plot for Day 15 measurement against Day 0. shRNA barcode reads for all samples from all time points were first normalized to 2×10⁷ reads. Values for triplicate samples were averaged. At Day 15, strong repression of shRNA expression in the absence of Doxycycline is evident in the correlations with the Day 0 reference. Plot of log10 mean normalized reads for Day 0 against Day 15 (Pearson correlation: R = 0.99).