Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

Stephanie Ibrahim; Catherine Robert; Camille Humbert; Clotilde Ferreira; Gwena&#235;lle Collod; Sonia Stefanovic

doi:10.3791/65328

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Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

JoVE Journal
Developmental Biology

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JoVE Journal Developmental Biology

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

2,160 Views

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10:03 min

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May 12, 2023

DOI:

10.3791/65328-v

DOI:

10.3791/65328-v

•

10:03 min

May 12, 2023

•

9 Views

Stephanie Ibrahim¹, Catherine Robert², Camille Humbert², Clotilde Ferreira¹, Gwenaëlle Collod², Sonia Stefanovic¹

¹Center for Cardiovascular Disease & Nutrition, U1263,Aix-Marseille University, ²Marseille Medical Genetics, U1251,Aix-Marseille University

Transcript

The main objective of this protocol is to study the interaction of genetic and environmental factors in the context of heart development and their contribution to congenital heart defects. Single nuclei approaches such as single nuclei RNA-seq and single nuclei ataxic are emerging as key methods to study cellular heterogeneity in health and disease during heart development. One limiting step of these experiments is that all the sample have to be run simultaneously.

While working with all the experimental conditions, collecting fresh enough material is for all the condition simultaneously could be challenging. Although there have been a significant progress in the field in single cell for recent years, the main difficulty is processing free samples. Avoiding the difficulty is extremely benefit to identify the molecular mechanism underlying congenital heart disease defects.

This protocol describes the steps to follow for successfully isolating high quality nuclei from frozen cardiac cell suspension obtained from mouse embryos. And our protocol is compatible with downstream single nuclei RNA-seq and single nuclei ataxic. We hope that this procedure will help interested researchers and encourage them to use this powerful method for their research.

Summary

Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses.