Adipocyte-Specific ATAC-Seq with Adipose Tissues Using Fluorescence-Activated Nucleus Sorting

Summary

We present a protocol for assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) specifically on adipocytes using nucleus sorting with adipose tissues isolated from transgenic reporter mice with nuclear fluorescence labeling.

Abstract

Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) is a robust technique that enables genome-wide chromatin accessibility profiling. This technique has been useful for understanding the regulatory mechanisms of gene expression in a range of biological processes. Although ATAC-seq has been modified for different types of samples, there have not been effective modifications of ATAC-seq methods for adipose tissues. Challenges with adipose tissues include the complex cellular heterogeneity, large lipid content, and high mitochondrial contamination. To overcome these problems, we have developed a protocol that allows adipocyte-specific ATAC-seq by employing fluorescence-activated nucleus sorting with adipose tissues from the transgenic reporter Nuclear tagging and Translating Ribosome Affinity Purification (NuTRAP) mouse. This protocol produces high-quality data with minimal wasted sequencing reads while reducing the amount of nucleus input and reagents. This paper provides detailed step-by-step instructions for the ATAC-seq method validated for the use of adipocyte nuclei isolated from mouse adipose tissues. This protocol will aid in the investigation of chromatin dynamics in adipocytes upon diverse biological stimulations, which will allow for novel biological insights.

Introduction

Adipose tissue, which is specialized for storing excess energy in the form of lipid molecules, is a key organ for metabolic regulation. The strict control of adipocyte formation and maintenance is vital for adipose tissue function and whole-body energy homeostasis¹. Many transcriptional regulators play a critical role in the control of adipocyte differentiation, plasticity, and function; some of these regulators are implicated in metabolic disorders in humans^2,3. Recent advances in high-throughput sequencing techniques for gene expression and epigenomic analysis have further facilitated....

Protocol

Animal care and experimentation were performed according to procedures approved by the Institutional Animal Care and Use Committee of Indiana University School of Medicine.

1. Preparations before beginning the experiment

1. Tissue preparation
   1. For adipocyte nucleus labeling, cross NuTRAP mice with adipocyte-specific adiponectin-Cre lines (Adipoq-Cre) to generate Adipoq-NuTRAP mice, which are hemizygous for both Adipoq-Cre and NuTRAP.
   2. Dissect the adipose tissues of interest from the Adipoq-NuTRAP mice as described previously¹⁴.
   3. Snap-freeze the tissues in liqui....

Results

To analyze adipose tissue using this ATAC-seq protocol, we generated Adipoq-NuTRAP mice that were fed chow diets; we then isolated adipocyte nuclei from epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT), and brown adipose tissue (BAT) by using flow cytometry. The isolated nuclei were used for tagmentation, followed by DNA purification, PCR amplification, quality check steps, sequencing, and data analysis, as described above. The purpose of this representative experiment was to profile the chrom.......

Discussion

In this paper, we have presented an optimized ATAC-seq protocol to assess adipocyte-specific chromatin accessibility in vivo. This ATAC-seq protocol using the Adipoq-NuTRAP mouse successfully generated adipocyte-specific chromatin accessibility profiles. The most critical factor for successful and reproducible ATAC-seq experiments is nucleus quality. It is critical to immediately snap-freeze the dissected adipose tissues in liquid nitrogen and store them safely at −80 °C without thawing until use. It .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Animals
Adiponectin-Cre mouse	The Jackson Laboratory	28020
NuTRAP mouse	The Jackson Laboratory	29899
Reagents & Materials
1.5 mL DNA-LoBind tubes	Eppendorf	86-923
100 µm cell strainer	Falcon	352-360
15 mL tubes	VWR	525-1071
2x TD buffer	Illumina	15027866
384-well PCR plate	Applied biosystem	4483285
40 µm cell strainer	Falcon	352-340
50 mL tubes	VWR	525-1077
AMPure XP reagent (SPRI beads)	Beckman Coulter	A63881
Bioanalyzer High Sensitivity DNA kit	Agilent Technologies	5067-4626
Clear adhesive film	Applied biosystem	4306311
DNase/RNase-free distilled water	Invitrogen	10977015
Dounce tissue grinder	DWK Life Sciences	357542
DTT	Sigma	D9779
DynaMag-96 side skirted magnet	Thermo Fishers	12027
FACS tubes	Falcon	28719128
HEPES	Boston BioProducts	BBH-75
Hoechst 33342	Invitrogen	2134015
KCl (2 M)	Boston BioProducts	MT-252
Magnetic separation rack for PCR 8-tube strips	EpiCypher	10-0008
MgCl2 (1 M)	Boston BioProducts	MT-200
MinElute PCR purification kit	Qiagen	28004
NEBNext High-Fidelity 2x PCR master mix	BioLabs	M0541S
NP40	Thermo Fishers	28324
PCR 8-tube strip	USA scientific	1402-4708
Protease inhibitor cocktail (100x)	Thermo Fishers	78439
Qubit dsDNA HS assay kit	Invitrogen	Q32851
Sucrose	Sigma	S0389-1KG
SYBR Green I (10,000x)	Invitrogen	S7563
TDE I enzyme	Illumina	15027865
Instruments
Flow cytometer	BD Biosciences	FACSAria Fusion
Qubit fluorometer	Invitrogen	Q33226
Real-Time PCR system	Thermo Fishers	QuantStudio?5