Identification of RNAi screen hits (published in Mejhert et al. 2020)
CellProfiler (Carpenter et al., 2006) was used to extract information from the RNAi screen images. For each extracted image feature, the median rz-score was calculated per gene, and the resulting matrix was filtered to exclude rows with a median |cell count rz-score| > 3 (n=547). Based on this dataset, image feature replicates were compared pair-wise across the genome-wide screen and non-reproducible parameters (median r-value < 0.3) were excluded. After this, a correlation matrix was generated by correlating all included image features with each other and the dimensionality of the matrix was tested using hierarchical clustering. Starting from the most reproducible image parameter in each major dimension (from the clustering), features were excluded if they covaried (|rho|>0.7) with the selected parameters and displayed low median reproducibility (r-value<0.4). This generated a dataset containing 21 high-confidence image parameters from which hits were identified. As the rz-scores for the different features varied in symmetry and dispersion (results not shown), a ranked-based approach was applied in which each image parameter was ranked from top to bottom by their respective rz-score, and hits were selected if they were distributed top or bottom 15 for one image parameter and/or top or bottom 50 for more than one image parameter. This resulted in 558 hits.
Identification of LD proteins (published in Mejhert et al. 2020)
Proteomics was performed on paired samples of intact cells and isolated lipid droplets. Fold changes comparing LD fractions with total cell lysates were based on label-free quantification. To calculate 99% confidence intervals for canonical LD proteins, the top 50 high-confidence proteins targeting to LDs were extracted from a previously published study (Bersuker et al., 2018) and overlapped with the results generated within this study.
Identification of LD proteins in the mouse liver proteome (published in Krahmer et al. 2018)
See this page for experimental details.
Identification of LD proteins in U2OS and Huh7 cells
Experimental details were published in Bersuker et al., 2018.