Human U2OS cells – RNAi Cell Painting experiment

Accession number BBBC025 · Version 1

Morphological profiles of RNAi-induced gene knockdown are highly reproducible but dominated by seed effects

Abstract: RNA interference and morphological profiling - the measurement of thousands of phenotypes from individual cells by microscopy and analysis image - are a potentially powerful combination. We show that morphological profiles of RNAi-induced knockdown using the Cell Painting assay are in fact highly sensitive and reproducible. However, we find that the magnitude and prevalence of off-target effects via the RNAi seed-based mechanism make morphological profiles of RNAi reagents targeting the same gene look no more similar than reagents targeting different genes. Pairs of RNAi reagents that share the same seed sequence produce image-based profiles that are much more similar to each other than profiles from pairs designed to target the same gene, a phenomenon previously observed in small-scale gene-expression profiling experiments. Various strategies have been used to enrich on-target versus off-target effects in the context of RNAi screening where a narrow set of phenotypes are measured, mostly based on comparing multiple sequences targeting the same gene; however, new approaches will be needed to make RNAi morphological profiling (that is, comparing multi-dimensional phenotypes) viable. We have shared our raw data and computational pipelines to facilitate research.


The images are of U2OS cells treated with each of 315 unique shRNA sequences and labeled with six labels that characterize eight organelles (the “Cell Painting” assay). The resolution of each image is 0.656 μm/pixel.

The images are now part of the Cell Painting Gallery under the dataset name "cpg0018-singh-seedseq".

Images can be accessed at this link 

Visit the Cell Painting Gallery for access instructions. 



CC0To the extent possible under law, Anne Carpenter has waived all copyright and related or neighboring rights to BBBC025v1. This work is published from: United States.