Protocol to identify genes required for cardiomyocyte development using Perturb-Seq.

Sushama Sivakumar; Lei Wang; Huan Zhao; Mpathi Nzima; David C Park; Yihan Wang; Anjana Sundarrajan; Chikara Takeuchi; Minnie Deng; W Lee Kraus; Gary C Hon; Nikhil V Munshi

doi:10.1016/j.xpro.2026.104790

PubMed · 42647169

Protocol to identify genes required for cardiomyocyte development using Perturb-Seq.

Sushama Sivakumar·Lei Wang·Huan Zhao·Mpathi Nzima·David C Park·Yihan Wang·Anjana Sundarrajan·Chikara Takeuchi·Minnie Deng·W Lee Kraus·Gary C Hon·Nikhil V Munshi

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Abstract

While Perturb-Seq combines CRISPR-based screening with single-cell RNA sequencing (scRNA-seq), large-scale experiments are costly and its application during development is complicated by differentiation heterogeneity. Here, we present a protocol to identify genes required for cardiomyocyte development using Perturb-Seq. We describe steps for sgRNA (single guide RNA) library cloning and infection, cardiomyocyte differentiation, cell hashing, super loading, and scRNA-seq. We then detail procedures for sequencing, mapping, and data analysis. For complete details on the use and execution of this protocol, please refer to Sivakumar et al.1.

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CRISPR Cell Differentiation Gene Expression Genomics High Throughput Screening Molecular Biology RNAseq Sequencing Stem Cells Journal Article

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Sushama Sivakumar, Lei Wang, Huan Zhao, Mpathi Nzima, David C Park, Yihan Wang, Anjana Sundarrajan, Chikara Takeuchi, Minnie Deng, W Lee Kraus, Gary C Hon, Nikhil V Munshi. 2026-08-25. Protocol to identify genes required for cardiomyocyte development using Perturb-Seq.. https://doi.org/10.1016/j.xpro.2026.104790

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