Tertiary siRNA screen (Iterative Indirect Immunofluorescence Imaging - 4i)
Description
Iterative indirect immunofluorescence experiment with EU metabolic labeling across a panel of 63 siRNA conditions in addition to control wells (tertiary siRNA screen), as described in Berry et al., 2022. Experimental conditions in duplicate. Single-cell features provided as background-subtracted mean and sum intensity values for the nucleus, cytoplasm, or whole-cell, as appropriate, for each fluorescence channel. Data cleanup and correction applied as described in the manuscript. Summaries provided on a per-well basis.
Files
Steps to reproduce
900 HeLa cells were plated per well in 384-well plates for reverse transfection onto a mixture of pooled siRNAs dispensed using an acoustic dispenser and Lipofectamine RNAiMAX (0.08µl per well in 10µL OptiMEM) according to manufacturer’s specifications. Cells were subsequently grown for 72 hours at 37°C in a final volume of 40µL growth media, to establish efficient knockdown of the targeted genes. 4i was performed as previously described {Gut.2018} with two modifications: Intercept blocking buffer (LI-COR Biosciences) was used for all blocking, primary and secondary antibody incubations, and 50mM HEPES was included in imaging buffer – which was then adjusted to a pH of 7.4. Before 4i experiments, all antibodies were tested for compatibility with elution buffer using the following criteria: similar staining on normal and elution-buffer treated cells, minimal residual signal after elution and re-staining with secondary antibody. After 4i, we also validated that agreement between replicates was high (Figure S8E) and that the technical variability between control (scrambled siRNA) wells was much less than the differences induced by the perturbations (Figure S8F). To ensure successful antibody elution in each cycle, we included elution controls in each imaging cycle. This consists of re-probing a test well with a secondary antibody in the imaging cycle after it was stained with primary and secondary antibody and imaged. Efficient elution was verified in all cases. Samples were imaged on an automated spinning-disk microscope (CellVoyager 7000, Yokogawa), which is equipped with four excitation lasers (405, 488, 568, 647nm) and two Neo sCMOS cameras (Andor). A 20X/NA0.75 objective was used. Computational image analysis was used to segment cells and nuclei from max-projected images, and intensity features calculated for all channels in the nucleus, cytoplasm and cell. Mitotic/apoptotic cells were identified and removed using iterative supervised machine learning in tissuemaps.