Figure S3.
Screens for nuclear size regulators. (a) sgRNA transduction results in cells with higher BFP intensity, and negative control sgRNAs do not affect nuclear size after viral infection. Two negative control sgRNAs were designed to have no target sites in the human genome. Cells (hTERT-RPE1 dCas9-KRAB-BFP PA-mCherry H2B-mGFP) underwent viral transduction and puromycin selection for 3 d before imaging. Both wild-type (WT) cells without viral transduction (gray dots), and cells infected with negative control sgRNAs (red dots) were seeded into a 96-well glass-bottom imaging dish. Images were collected for cells with/without sgRNA viral transduction, and both nuclear size and mean BFP intensity of each nucleus were analyzed using the Auto-PhotoConverter μManager plugin (number of wild-type cells analyzed = 2,756; number of negative control sgRNA infected cells analyzed = 5,653). Besides the BFP expressed from the dCas9 construct, another BFP was encoded on the sgRNA construct, and higher BFP intensity was used as a marker for successful infection. The boundary measured from comparison between sgRNA-infected cells and wild-type cells: ln(mean BFP intensity) = 7.6 was also used as a threshold to determine which cells were successfully transduced with sgRNA. Analysis from imaging data shows no correlation between nuclear size and BFP intensity. (b) eFDR-η curve for screening result shown in Fig. 4 c. A score η summarizing effects from both severity of the phenotype (phenotypic score) as well as trustworthiness of the phenotype [−ln(P value)] was calculated for each gene and simulated negative control. The optimal cutoff for score η (red dotted line) was determined by calculating an eFDR at multiple values of η as the number of simulated negative controls with score η higher than the cutoff (false positives) divided by the sum of genes and simulated negative controls with score η higher than the cutoff (all positives). The cutoff score η resulting in an eFDR of 0.1% (black dotted line) was used to call hits for further analysis. An example analysis is described in detail in Data S5. (c) Screening result of the other replicate shown in volcano plot and its corresponding eFDR-η curve as described in Fig. S3 b. (d) Number of hits identified using data averaging using different numbers of runs and/or different library compositions. Error bar: SD.

Screens for nuclear size regulators. (a) sgRNA transduction results in cells with higher BFP intensity, and negative control sgRNAs do not affect nuclear size after viral infection. Two negative control sgRNAs were designed to have no target sites in the human genome. Cells (hTERT-RPE1 dCas9-KRAB-BFP PA-mCherry H2B-mGFP) underwent viral transduction and puromycin selection for 3 d before imaging. Both wild-type (WT) cells without viral transduction (gray dots), and cells infected with negative control sgRNAs (red dots) were seeded into a 96-well glass-bottom imaging dish. Images were collected for cells with/without sgRNA viral transduction, and both nuclear size and mean BFP intensity of each nucleus were analyzed using the Auto-PhotoConverter μManager plugin (number of wild-type cells analyzed = 2,756; number of negative control sgRNA infected cells analyzed = 5,653). Besides the BFP expressed from the dCas9 construct, another BFP was encoded on the sgRNA construct, and higher BFP intensity was used as a marker for successful infection. The boundary measured from comparison between sgRNA-infected cells and wild-type cells: ln(mean BFP intensity) = 7.6 was also used as a threshold to determine which cells were successfully transduced with sgRNA. Analysis from imaging data shows no correlation between nuclear size and BFP intensity. (b) eFDR-η curve for screening result shown in Fig. 4 c. A score η summarizing effects from both severity of the phenotype (phenotypic score) as well as trustworthiness of the phenotype [−ln(P value)] was calculated for each gene and simulated negative control. The optimal cutoff for score η (red dotted line) was determined by calculating an eFDR at multiple values of η as the number of simulated negative controls with score η higher than the cutoff (false positives) divided by the sum of genes and simulated negative controls with score η higher than the cutoff (all positives). The cutoff score η resulting in an eFDR of 0.1% (black dotted line) was used to call hits for further analysis. An example analysis is described in detail in Data S5. (c) Screening result of the other replicate shown in volcano plot and its corresponding eFDR-η curve as described in Fig. S3 b. (d) Number of hits identified using data averaging using different numbers of runs and/or different library compositions. Error bar: SD.

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