a, Scatter plot illustrating the correlation of normalized reads per shRNA at all three time points (T0, T1 and T2; top to bottom) compared to the plasmid pool in all three independent replicates (left to right). The schematic to the right illustrates the different sampling time points subjected to deep sequencing. Top hits enriched under JQ1 treatment and positive controls from the initial negative-selection screen (T1) are marked with coloured dots according to the legend on the right. b, Pooled negative-selection screening depicting changes in representation of 2,917 shRNAs during 7 days of culture. shRNA fold depletion values were calculated by dividing the number of reads after 7 days of culture (T1) by the number of reads obtained from the plasmid pool, and are plotted as the mean of three replicates in ascending order. Completely depleted shRNAs (0 reads at T1) obtained a fold depletion value of 1 × 10−3. Positive control shRNAs targeting essential genes are marked in red; negative control shRNAs are depicted in green. c, Scatter plot depicting all genes ranked by the sum of their average depletion score of all shRNAs across all three replicates. Top scoring hits were defined as genes for which at least two shRNAs showed an average depletion of eightfold after 7 days of shRNA expression and are marked in red (45 genes). d, IC50 determination for JQ1 in murine MLL–AF9;NrasG12D AML cells (RN2). Obtained numbers of viable cells per ml were normalized to DMSO (n = 3, mean ± s.e.m.). e, Table showing the top ten enriched shRNAs at T2. shRNAs targeting Suz12, Dnmt3a and Psip1 are strongly enriched in all three independent replicates. f, Relative mRNA abundance (RPKM) of shRNA target genes in JQ1-resistant leukaemia cells expressing the indicated shRNAs, plotted relative to leukaemia cells expressing Ren.713.