Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

Summary

A novel protocol for the preparation of chromatin from adult mouse skeletal muscle adapted to the study of gene regulation in muscle fibers by chromatin immunoprecipitation is presented.

Abstract

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a high content of structural proteins. Dissected limb muscles from adult mice are physically disrupted by mechanical homogenisation, or a combination of mincing and douncing, in a hypotonic buffer before formaldehyde fixation of the cell lysate. The fixed nuclei are purified by further cycles of mechanical homogenisation or douncing and sequential filtrations to remove cell debris. The purified nuclei can be sonicated immediately or at a later stage after freezing. The chromatin can be efficiently sonicated and is suitable for chromatin immunoprecipitation experiments, as illustrated by the profiles obtained for transcription factors, RNA polymerase II, and covalent histone modifications. The binding events detected using chromatin prepared by this protocol are predominantly those taking place in the muscle fiber nuclei despite the presence of chromatin from other fiber-associated satellite and endothelial cells. This protocol is therefore adapted to study gene regulation in the adult mouse skeletal muscle.

Introduction

Chromatin immunoprecipitation (ChIP) coupled to quantitative polymerase chain reaction (qPCR) and high throughput sequencing (ChIP-seq) have become the methods of choice to study transcription and epigenetic regulation of gene expression in various tissues and cell-types¹. This technique allows genome-wide profiling of covalent chromatin modifications, histone variant occupancy, and transcription factor binding^2,3.

While performing ChIP from cultured cells is well established, ChIP from mammalian tissues remains more challenging. Preparing chromatin for ChIP ....

Protocol

Mice were kept in accordance with the institutional guidelines regarding the care and use of laboratory animals and in accordance with National Animal Care Guidelines (European Commission directive 86/609/CEE; French decree no.87-848). All procedures were approved by the French national ethics committee.

1. Isolation of Muscle Tissue

1. Sacrifice one adult 6 to 8-week-old mouse by cervical dislocation. Sterlise the limb by rinsing it with 70% ethanol and dissect the hind limb muscles (Gastrocnemius, Tibialis Anterior, Quadriceps) using fine point scissors and forceps.
   NOTE: One mouse should yield around 500 mg of tissue.

Results

To isolate nuclei, we performed mechanical homogenization of the dissected and minced muscle tissue for either 15 or 45 s, at 18,000 and 22,000 rpm (see Table of Materials). In all conditions, nuclei could be separated from the tissue debris, but the yield was optimal using the lower speed (Figure 1A-B). Nuclei could also be prepared by douncing for 3 - 5 min (Figure 1A, and data not shown), but .......

Discussion

Here we describe a novel protocol for preparing chromatin from adult mouse skeletal muscles and show that this chromatin is suitable for ChIP experiments that detect transcription factor binding and covalent histone modifications in the muscle fiber nuclei. This protocol involves several critical steps. The first is tissue disruption that can be performed either by dounce or by mechanical shearing. Mechanical shearing is faster and more reproducible, and is therefore the method of choice. Nevertheless, if no suitable app.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
T 25 digital ULTRA-TURRAX	T 18 ULTRA-TURRAX	0009022800
PMSF	SIGMA-ALDRICH CHIMIE SARL	P7626-25G
Protease Inhibitor Cocktail-EDTA Free	Roche Diagnostics	11873580001
Protein G Sepharose beads	SIGMA-ALDRICH CHIMIE	P-3296
Proteinase K	SIGMA-ALDRICH CHIMIE	P-2308
Cell Strainers 70um	Corning BV	431751
Cell Strainers 40um	Corning BV	352340
Formaldehyde EM grade	Euromedex	15710-S
Rnase A	Fischer Scientific	12091039
Phenol:Chloroform:Isoamyl Alcohol 25:24:1	SIGMA-ALDRICH CHIMIE	P3803
BSA	SIGMA-ALDRICH CHIMIE	B4287-25G
yeast tRNA	SIGMA-ALDRICH CHIMIE	R5636
Glycogen Blue	AMIBION	AM9516
Pol II ChIP Antibody	Santa Cruz	SC-9001
H3K27ac ChIP Antibody	Active Motif	39133
Tead4 ChIP Antibody	Aviva Systems Biology	(ARP38276_P050)
IGEPAL	SIGMA-ALDRICH CHIMIE	I-3021
E220 Focused Ultrasonicator	Covaris E220
Name	Company	Catalog Number	Comments
Additional items
Scissors
Loose Dounce
15 ml and 50 ml falcon tubes
14 ml round bottom tubes
1.5 ml and 2 ml Eppendorf tubes
70 μm and 40 μm cell strainers (Corning)