Protocol to identify genes required for cardiomyocyte development using Perturb-Seq
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
Authors
- Sushama Sivakumar (ORCID: https://orcid.org/0000-0001-7877-4821)
- Anjana Sundarrajan
- Minnie Deng
- Huan Zhao
- Gary C. Hon (ORCID: https://orcid.org/0000-0002-1615-0391)
- Chikara Takeuchi (ORCID: https://orcid.org/0000-0001-6855-6580)
- Yihan Wang (ORCID: https://orcid.org/0000-0001-7440-417X)
- Nikhil Munshi (ORCID: https://orcid.org/0000-0002-8397-100X)
- Mpathi Nzima
- Lei Wang
- David C. Park
- W. Lee Kraus
Institutions
- Southwestern Medical Center (US)
- The University of Texas Southwestern Medical Center (US)
Publication Details
- Journal
- STAR Protocols
- Published
- 2026-08-25
- DOI
- https://doi.org/10.1016/j.xpro.2026.104790
- Primary Topic
- Congenital heart defects research
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- Burroughs Wellcome Fund
- National Heart, Lung, and Blood Institute
- National Human Genome Research Institute
- National Institute of General Medical Sciences