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Experimental and Computational Approaches for Single-Cell Enhancer Perturbation Assay

  • Shiqi Xie
  • Gary C. HonEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1935)

Abstract

Transcriptional enhancers drive cell-type-specific gene expression patterns, and thus play key roles in development and disease. Large-scale consortia have extensively cataloged >one million putative enhancers encoded in the human genome. But few enhancers have been endogenously tested for function. For almost all enhancers, it remains unknown what genes they target and how much they contribute to target gene expression. We have previously developed a method called Mosaic-seq, which enables the high-throughput interrogation of enhancer activity by performing pooled CRISPRi-based epigenetic suppression of enhancers with a single-cell transcriptomic readout. Here, we describe an optimized version of this method, Mosaic-seq2. We have made several key improvements that have significantly simplified the library preparation process and increased the overall sensitivity and throughput of the method.

Key words

Single-cell RNA-seq Enhancer CRISPRi Single-cell perturbation 

Notes

Acknowledgments

This work is supported by the Cancer Prevention Research Institute of Texas (CPRIT) (RR140023, G.C.H.), NIH (DP2GM128203, G.C.H.), the Department of Defense (PR172060, G.C.H.), the Welch Foundation (I-1926-20170325, G.C.H.), and the Green Center for Reproductive Biology. S.X. is an American Heart Association fellow (16POST29910007).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Obstetrics and Gynecology, Cecil H. and Ida Green Center for Reproductive Biology SciencesUniversity of Texas Southwestern Medical CenterDallasUSA

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