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

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.

Abstract

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

Introduction

Among birth defects, congenital heart defects (CHDs) are the most common, occurring in about 1% of live births each year^1,2. Genetic mutations are identified in only a minority of cases, implying that other causes, such as abnormalities in gene regulation, are involved in the etiology of CHD^2,3. Cardiac development is a complex process of diverse and interacting cell types, making the identification of causal noncoding mutations and their effects on gene regulation challenging. Organogenesis of the heart begins with cellular progenitors that give rise ....

Protocol

The animal procedure adopted in this study was approved by the animal ethics committees of the Aix-Marseille University (C2EA-14) and was carried out according to protocols approved by the appointed national ethical committee for animal experimentation (Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche; Authorization Apafis N°33927-2021111715507212).

1. Setting up the timed mating prior to dissection

1. To genera.......

Discussion

The analysis of the cellular composition of the developing heart by combined snRNA-seq and snATAC-seq studies provides a deeper understanding of the origin of congenital heart disease²⁶. Several research laboratories have studied the effects of cardiac tissue cryopreservation on snRNA-seq²⁷. Conducting snRNA-seq and snATAC-seq using fresh micro-dissected tissue from mouse models of human disease can be logistically challenging when comparing different experimental condition.......

Materials

Name	Company	Catalog Number	Comments
2100 Bioanalyzer Instrument	Agilent		No catalog number
5M Sodium chloride (NaCl)	Sigma	59222C-500ML
BSA 10%	Sigma	A1595-50ML
Chromium Next GEM Chip J Single Cell Kit, 16 rxns	10X Genomics	1000230
Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle, 4 rxns (including Nuclei Buffer 20X)	10X Genomics	1000285
Countess cell counting chamber slides	Invitrogen	C10283
Countess III FL	Thermofisher		No catalog number
Digitonin (5%)	Thermofisher	BN2006
DMSO	Sigma	D2650-5x5ML
DNA LoBind Tubes	Eppendorf	22431021
D-PBS	Thermofisher	14190094	Sterile and RNase-free
Dual Index Kit TT Set A 96 rxns	10X Genomics	1000215
Falcon 15 mL Conical Centrifuge Tubes	Fisher Scientific	352096
Falcon 50 mL Conical Centrifuge Tubes	Fisher Scientific	10788561
HI-FBS	Thermofisher	A3840001	Heat inactivated
High sensitivity DNA kit	Agilent	5067-4626
Igepal CA-630	Sigma	I8896-50ML
LIVE/DEAD Viability/Cytotoxicity Kit	Thermofisher	L3224
MACS Dead Cell Removal kit: Dead Cell Romoval MicroBeads, Binding Buffer 20X	Miltenyi Biotec	130-090-101
MACS SmartStrainers (30 µm)	Miltenyi Biotec	130-098-458
Magnesium chloride (MgCl2)	Sigma	M1028-100ML
Milieu McCoy 5A	Thermofisher	16600082
MS Columns	Miltenyi Biotec	130-042-201
NovaSeq 6000 S2	Illumina		No catalog number
Penicillin Streptomycin (Pen/Strep)	Thermofisher	15070063
PluriStrainer Mini 40µm	PluriSelect	V-PM15-2021-12
Rock inhibitor	Enzo Life Sciences	ALX-270-333-M005
Single Index Kit N Set A, 96 rxn	10X Genomics	1000212
Standard 90mm Petri dish Sterilin	Thermofisher	101R20
Sterile double-distilled water	Thermofisher	R0582
Trizma Hydrochloride solution (HCl)	Sigma	T2194-100ML
Trypan Blue stain (0.4%)	Invitrogen	T10282
Trypsin 0.05% - EDTA 1X	Thermofisher	25300054
Tween20	Sigma	P9416-50ML
Wide orifice filtered pipette tips 200 μl	Labcon	1152-965-008-9
ZEISS SteREO Discovery.V8	ZEISS		No catalog number