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Updated: Jun 27, 2026

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Published on: May 29, 2014

Benchmarking and optimizing Perturb-seq in differentiating human pluripotent stem cells.

Sushama Sivakumar¹, Yihan Wang², Sean C Goetsch¹

¹Department of Internal Medicine, Division of Cardiology, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Biorxiv : the Preprint Server for Biology

|February 3, 2025

Summary

This summary is machine-generated.

Perturb-seq enables studying gene and enhancer functions in human stem cells. This research optimized the technique for large-scale use, revealing gene networks in cardiomyocyte development.

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Area of Science:

Genomics
Stem Cell Biology
Molecular Biology

Background:

Perturb-seq is a valuable tool for understanding gene and enhancer functions in biological pathways.
Technical hurdles have previously restricted its use in stem cell research.
The Impact of Genomic Variants on Function (IGVF) consortium aims to generate large-scale genomic datasets.

Purpose of the Study:

To benchmark Perturb-seq across various CRISPRi methods, genomic targets, and human pluripotent stem cell types.
To optimize a cost-effective protocol for large-scale Perturb-seq data generation.
To identify shared regulatory networks involved in stem cell differentiation and disease.

Main Methods:

Benchmarking of multiple CRISPR interference (CRISPRi) modalities.
Application across diverse genomic targets and human pluripotent stem cells.
Directed differentiation into multiple cell lineages with various sgRNA delivery systems.
Development of a dynamic quality assessment for long-term experiments.
Analysis of 1,996,260 sequenced cells.

Main Results:

Established an optimized and cost-effective Perturb-seq protocol for large-scale studies.
Identified shared regulatory networks connecting disease-associated elements to downstream targets during cardiomyocyte differentiation.
Demonstrated the utility of Perturb-seq across multiple cell types and differentiation protocols.

Conclusions:

The optimized Perturb-seq protocol overcomes previous technical limitations for stem cell applications.
This work provides open tools and resources for functional genomics research.
The findings offer insights into gene regulatory networks critical for development and disease.