Teton Atlas™ with Direct In Sample Sequencing, Optical Pooled Screening (A549 cells)

Background 

This study demonstrates the application of Direct In Sample Sequencing (DISS) for optical pooled screening (OPS) in A549 lung epithelial cells. By integrating DISS into the OPS workflow, we directly sequenced perturbation identities within the same cells used for imaging, linking CRISPR-induced changes to phenotypic and protein-level readouts. This approach enables high-content, multiplexed analysis of how inflammation, Wnt, and TGF pathway perturbations shape cellular responses under IL-1 stimulation across a 48-hour time course. More information about the full experiment can be found in our pre-print and our 5D Multiomics webinar. 

Methods  

A549 Cas9-expressing cells were transfected with a 28-guide CRISPR library targeting CTNNB1, GSK3B, IL1R1, SMAD7, and TGFBR2, along with OR1I1 (olfactory receptor; no expected phenotype in A549) and non-targeting controls. Cells were profiled at baseline and following IL-1 stimulation at 30 mins, 1 hr, 8 hrs, 24 hrs, and 48 hrs. Guide sequencing, cell paint, and protein detection was performed using the Teton Atlas Low Output workflow with the MAPK protein panel.   

Results  

Across the experiment, we studied approximately 228,000 single cells, providing broad coverage of each perturbation and condition. On average, we profiled 26,000 cells per targeted gene, corresponding to roughly 4,000 cells per gene per condition across the six time points. This dataset captures how CRISPR perturbations alter cytokine-driven signaling over time.  

Instructions 

Fill out the form below to download the dataset and Cell Montage file referenced in the video below.