A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation

Summary

We present a filtration-based protocol to isolate high-quality nuclei from cross-linked mouse skeletal muscle wherein we removed the need for ultracentrifugation, making it easily applicable. We show that chromatin prepared from the nuclei is suitable for chromatin immunoprecipitation and likely chromatin immunoprecipitation sequencing studies.

Abstract

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a challenge for ChIP due to technical difficulty in isolation of high-quality nuclei with minimal contamination of myofibrils. Previous protocols have attempted to purify nuclei before cross-linking, which incurs the risk of altered DNA-protein interaction during the prolonged nuclei preparation process. In the current protocol, we first cross-linked the skeletal muscle tissue collected from mice, and the tissues were minced and sonicated. Since we found that ultracentrifugation was not able to separate nuclei from myofibrils using cross-linked muscle tissue, we devised a sequential filtration procedure to obtain high-quality nuclei devoid of significant myofibril contamination. We subsequently prepared chromatin by using an ultrasonicator, and ChIP assays with anti-BMAL1 antibody revealed robust circadian binding pattern of BMAL1 to target gene promoters. This filtration protocol constitutes an easily applicable method to isolate high-quality nuclei from cross-linked skeletal muscle tissue, allowing consistent sample processing for circadian and other time-sensitive studies. In combination with next-generation sequencing (NGS), our method can be deployed for various mechanistic and genomic studies focusing on skeletal muscle function.

Introduction

Skeletal muscle plays important roles in physiology and behavior. The multi-nucleated muscle fiber consists of myofibrils where actin and myosin form functional units called sarcomeres to generate contractile force. Skeletal muscle is also the largest metabolic organ in the body, accounting for >80% postprandial glucose intake and regulating insulin response and metabolic homeostasis^1,2. Muscle physiology and metabolism are closely regulated by the circadian clock, an intrinsic biological timer^3,4,5,

Protocol

Animal care was performed under Institutional Animal Care and Use Committee (IACUC) guidelines, and the procedures were conducted according to an animal protocol approved by the University of Texas Health Science Center at Houston.

1. Nuclei Isolation from Cross-linked Skeletal Muscle

1. Weigh and mince hind limb skeletal muscle, isolated from approximately 20-week old C57BL/6 male mice, in ice-cold phosphate buffered saline (PBS) as described in detail previously²².
   NOTE: We usually obtain 1.0 - 1.5 g of skeletal muscle from one mouse.
   1. Place minced skeletal muscle tissue in a 50-mL conica....

Results

Here we performed formaldehyde cross-linking immediately after tissue collection to preserve real-time DNA-protein interaction. However, we found that sucrose or colloidal gradient, commonly used for nuclei isolation^23,24, was not effective in separating nuclei from myofibrils (data not shown). The reason may be that cross-linking conferred similar gravity for nuclei and myofibrils. Therefore, we developed a serial filtration proc.......

Discussion

Here we describe a robust method where cross-linked skeletal muscle tissues were used to isolate high-quality nuclei. Sequential filtration was carried out to effectively separate nuclei from debris, and ultrasonic acoustic energy from dish-shaped transducer sheared the chromatin for ChIP analysis. The results showed circadian time-specific binding of BMAL1 to target promoters.

ChIP can be employed to capture real-time protein occupancy on genomic DNA when cross-linking takes place. To take ad.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Materials
Formaldehyde solution	Sigma Aldrich	F8775
Glycine	Fisher Scientific	BP381-5
Trypan Blue solution (0.4%)	Fisher Scientific	15250061
RNase A	Sigma Aldrich	10109142001
Protease K	Sigma Aldrich	3115887001
Chicken Anti-BMAL1 antibody			Generated in chicken (Cocalico Biologicals) against antigen aa 318 - 579, and IgY was affinity purified using the same antigen.
Chicken IgY Precipitating Resin	GenScript	L00405
Equipment
KINEMATICA Polytron PT2100 Benchtop Homogenizer	Fisher Scientific	08-451-178
15 mL Dounce tissue grinder	Whearton	357544
Falcon Cell Strainers 100 µm	Fisher Scientific	08-771-19
Falcon Cell Strainers 70 µm	Fisher Scientific	08-771-1
Falcon Cell Strainers 40 µm	Fisher Scientific	08-771-2
pluriStrainer 30 µm	PluriSelect	43-50030-03
pluriStrainer 20 µm	PluriSelect	43-50020-03
pluriStrainer 10 µm	PluriSelect	43-50010-03
Covaris S2 Focused-ultrasonicator	Covaris	Model S2
Labquake	Thermo Scientific	C415110
CCD Microscope Camera	Leica Microsystems	DFC3000 G
Reagent Kit
DNA extraction kit	Thermo Scientific	K0691
Buffers
All buffer components are discribed in the protocol. Each component was purchased from Sigma Aldrich