Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications

Summary

This publication describes a protocol for the isolation of nuclei from mature adipocytes, purification by fluorescence-activated sorting, and single-cell level transcriptomics.

Abstract

Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent pathways. Until recently, thermogenic adipocytes were considered a homogeneous population. However, recent studies have indicated that there are multiple subtypes or subpopulations that are distinct in developmental origin, substrate use, and transcriptome. Despite advances in single-cell genomics, unbiased decomposition of adipose tissues into cellular subtypes has been challenging because of the fragile nature of lipid-filled adipocytes. The protocol presented was developed to circumvent these obstacles by effective isolation of single nuclei from adipose tissue for downstream applications, including RNA sequencing. Cellular heterogeneity can then be analyzed by RNA sequencing and bioinformatic analyses.

Introduction

Studies have shown that brown adipose tissue (BAT) has a remarkable capacity to dissipate energy. Two types of thermogenic adipocytes with distinct developmental features exist in both rodents and humans: beige adipocytes and classical brown adipocytes. While classical brown adipocytes are located mostly in interscapular BAT depots, beige adipocytes sporadically emerge in white adipose tissue (WAT) in response to certain physiological cues, such as chronic cold exposure, a process referred to as "browning" or "beiging". Through the use of advanced imaging, it is now clear that adult humans have substantial depots of UCP1⁺ BAT, especially in ....

Protocol

Animal care and experimentation were performed according to procedures approved by the Institutional Animal Care and Use Committee at the Albert Einstein College of Medicine.

1. Preparation of tissue digestion and lysis buffers

1. Prepare tissue digestion buffer.
   1. Prepare ~1 mL of digestion buffer for every gram of adipose tissue.
   2. Weigh out 1.5 U/mL collagenase D and 2.4 U/mL dispase II and add phosphate buffered saline (PBS).
2. Prepare nuclei preparation buffer (NPB).
   1. Prepare 10 mM HEPES, 1.5 mM magnesium chloride, 10 mM potassium chloride, 250 mM sucrose, and 0.1% NP-40 in n....

Results

Unsorted adipocyte nuclei contain debris and doublets that create noise and high background in downstream single-cell RNA sequencing. The representative FACS gate strategy is shown in Figure 1. The nuclei were first selected based on forward scatter (FSC) and side scatter (SSC) (A), then, only singlets were selected based on the combination of width and heights of SSC (B). Finally, only DAPI-positive events were selected and .......

Discussion

A straightforward and robust method to isolate single nuclei and study adipose tissue heterogeneity is presented. Compared to whole tissue RNA sequencing, this workflow offers an unbiased view of cellular heterogeneity and population-specific markers. This is significant and innovative for the advancement of adipocyte biology, molecular metabolism, and obesity research.

This protocol is particularly optimized for downstream application of snRNA-seq. The "cleanup" step to achieve isolat.......

Materials

Name	Company	Catalog Number	Comments
autoMACS Rinsing Solution	Miltenyi Biotec	130-091-222	PBS with EDTA; sterile-filtered
BSA	Sigma	A1595
CaCl2	Sigma	21115
Cell filter 100 μm	Corning	431752
Cell filter 40μm	Corning	431750
CellTrics (30 μm)	Sysmex	04-004-2326
Collagenase D	Roche	11088866001
Countess II FL Automated Cell Counter	Invitrogen	AMQAF1000
DAPI	Sigma	D9542
Dispase II	Roche	4942078001
HEPES	Sigma	H4034
KCl	Fisher	P217-3
MACS SmartStrainers (30 µm)	Miltenyi Biotec	130-098-458	Stackable filters
MgCl2	Sigma	M1028
MoFloXDP Cell Sorter	Beckman Coulter	ML99030
NP-40	Sigma	74385
Protector RNase Inhibitor	Roche	3335402001
Sucrose	Fisher	S5-3