The present protocol summarizes a universal method for isolating, purifying, and upstream processing of murine white adipocytes optimized for downstream total RNA sequencing, Nuclei Extraction by SONication (NEXSON), and ChIP-seq.
Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type in white adipose tissue. Understanding how adipocytes function and respond to (epi)genetic and environmental signals is essential for identifying the cause(s) of obesity. RNA and chromatin have previously been isolated from adipocytes using enzymatic digestion. In addition, protocols have been developed for nuclear isolation, where purification is achieved by fluorescence-activated cell sorting (FACS) of adipocyte-specific transgenic reporters. One of the greatest challenges to achieving high yield and quality during such protocols is the substantial amount of lipid contained in adipose tissue. The present protocol describes an optimized procedure for isolating mature adipocytes that leverages heptane to separate lipids from the targets of interest (RNA/chromatin). The resulting RNA has high integrity and generates high-quality RNA-seq results. Likewise, the procedure improves nuclei yield rate and generates reproducible ChIP-seq results across samples. Therefore, the current study provides a reliable and universal murine adipocyte isolation protocol suitable for whole-genome transcriptome and epigenome studies.
Obesity is typically understood as a disease of excess fat accumulation that contributes to a heightened risk of type 2 diabetes, cardiometabolic disease, and several forms of cancers1,2,3. While the current understanding of obesity is heavily rooted in genetics (from both human and rodent studies), some 30%-70% of metabolic disease predisposition is non-genetic in origin4,5,6,7,8 and remains ill-defined.
Adipose tissue plays a critical role in obesity and other metabolic diseases9,10. Adipose tissue comprises mature adipocytes and the stromal vascular fraction, including preadipocytes, endothelial cells, and immune cells. It is still unclear how each cell type contributes to obesity and how adipocyte dysregulation contributes to obesity. Reproducible and effective isolation and purification protocols for mature adipocyte epigenome studies are of interest to the field.
Mature adipocytes have long been isolated for gene expression analyses11 and epigenome studies12,13. There are two main strategies for isolating adipocytes. The first is to use enzymatic digestion to separate the mature adipocytes from the rest of the cell types in the stromal vascular fraction11,14. The second is to dounce the adipose tissue to release intact nuclei and then recover the nuclei based on a fluorescent reporter by fluorescence-activated cell sorting (FACS)12,13, which requires specialized transgenic reporter models. The technical challenge in each case is that mature adipocytes contain high concentrations of lipids (Figure 1), which reduces the quality and/or yield of total RNA15,16 and nuclei17. Here, an optimized enzymatic digestion procedure is described for isolating mature adipocytes, in which the advantage of the heptane is to quickly and efficiently dissolve and remove lipids18 prior to RNA extraction or the nuclei isolation steps by Nuclei Extraction by SONication (NEXSON)19. The protocol ensures excellent recovery and quality of total RNA for genome-wide studies and significantly improves the yield of intact nuclei for reproducible ChIP-seq.
All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC), protocol number: 18-10-028. 12-week-old male C57BL/6J mice were euthanized with CO2 and dissected to collect the fat pads from the epididymal white adipose tissue (eWAT).
1. Adipocyte isolation
2. Total RNA extraction
NOTE: Perform this step in a chemical hood.
3. Adipocyte fixation for ChIP-seq
NOTE: Perform step 3. in a chemical hood.
4. Nuclei extraction and chromatin shearing
NOTE: This procedure is adapted from Arrigoni et al.19.
Adipocytes were isolated from six fat pads, the heptane-based RNA extraction was performed (step 2.), and the resulting RNA was analyzed on the automated electrophoresis instrument. The calculated RNA integrity number (RIN) for all samples was >8 (Figure 3A), indicating a high-quality and reproducible RNA preparation. The total RNA prep kit was then used to prepare RNA libraries, and each sample was sequenced on the next-generation sequencer to reach a read depth of ≥40 million reads. The Phred score for all samples (Figure 3B) and per sequence (Figure 3C) was ≥30, indicative of high-quality DNA sequences20. Thus, heptane removal supports the isolation of high-yield, high-quality RNA suitable for RNA sequencing.
In the formaldehyde fixation step, three heptane-containing nuclear isolation preparations and one control preparation without heptane were also performed. The sheared chromatin was then analyzed on the automated electrophoresis instrument. In both cases, the chromatin was sheared to a 100-800 bp size range (Figure 4A), ideal for downstream ChIP-seq procedures19. Importantly, ~5 times more chromatin was obtained from the heptane-treated samples (11.5 ng/µL, 9.42 ng/µL, and 9.6 ng/µL) relative to the untreated samples (2.2 ng/µL). H3K4me3 and H3K27me3 ChIP-seq were performed following Arrigoni et al.19. As shown in Figure 4B, the signal tracks from three independent heptane-treated samples were comparable to the track of the heptane-untreated sample for both histone marks. Heptane treatment does not interfere with the quality of ChIP-seq but significantly improves nuclei (and sheared chromatin) yield.
Figure 1: Isolated adipocytes. Isolated adipocytes were stained with DAPI (blue) to stain the nucleus and BODIPY (green) for the lipids. The cytosolic lipid droplets comprise up to 95% of the volume of the adipocytes and pose a technical challenge for achieving high yields during RNA and chromatin extractions. Scale bar = 100 µm. Please click here to view a larger version of this figure.
Figure 2: Schematic flow of the adipocyte isolation for transcriptome and epigenome analysis. The entire workflow is depicted, from adipocyte isolation to transcriptome or epigenome application. The key steps and representative results are shown. (A) The isolated adipocytes float on the top layer, separating from the stromal vascular fraction as a pellet at the bottom of the tube. (B) The use of heptane to remove lipids before RNA extraction with RNA isolation reagent. (C) The use of heptane to remove lipids during adipocyte fixation. (D) The representative image of isolated adipocyte nuclei must be intact and round. Scale bar = 20 µm. The scheme was created with BioRender.com. Please click here to view a larger version of this figure.
Figure 3: Representative electropherogram of RNA integrity and quality scores after RNA-seq. (A) Samples 1-6 represent six replicates of heptane-treated adipocytes, and their RNA integrity analysis was run on the automated electrophoresis instrument. The RNA integrity number (RIN) was based on 18S and 28S ribosomal RNA ratios and represents the RNA quality. Scale from 1 (much degraded) to 10 (the least degraded). (B,C) The sequencing read quality score was determined by multiQC analysis (B) for the mean quality score on bases (C) and for the quality score per sequence. Please click here to view a larger version of this figure.
Figure 4: Representative electropherogram of sheared chromatin and enrichment peaks from ChIP-seq. (A) Representative profiles from the automated electrophoresis instrument showing the chromatin size distributions of the control (sample 1, top left) and the heptane-treated adipocyte chromatin preparations (samples 2, 5, and 8, top right and bottom two). The chromatin concentrations in the final preparations are indicated on the top left of each image. (B) Genome browser screenshot made using the Integrative Genomics Viewer (IGV). The upper part of the plot shows H3K4me3 ChIP-seq profiles for three (red) heptane-treated samples and one (blue) control. The lower panel shows the same for the ChIP-seq of H3K27me3. Please click here to view a larger version of this figure.
Buffer | Composition |
Digestion buffer (for 3000 mg or less of fat pad/ 20 mL) | 20 mL of Dulbecco's Modified Eagle Medium (DMEM) |
0.3 g of fatty acid-free BSA | |
0.1 g collagenase type 2 | |
Farnham lab buffer | 5 mM PIPES (pH 8) |
85 mM KCl | |
0.5% IGEPAL | |
Shearing buffer | 10 mM Tris-HCl pH 8 |
0.1% SDS | |
1 mM EDTA |
Table 1: Composition of the different buffers used in the present study.
The adipocyte isolation protocol presented here is based on well-accepted enzymatic digestion methods11,14 to separate mature adipocytes (floating) from the white adipose tissue's remaining stromal vascular fraction. It provides a straightforward and universal approach to purifying mature adipocytes in any mouse model. As demonstrated above, the protocol is suitable for downstream use for whole transcriptome and ChIP-seq analyses. It provides sufficient yield to generate multiple epigenomic profiles (e.g., several histone modifications plus RNA-seq) from the same individual fat pad.
To ensure the high yields that enable the preparation of such matched epigenome data, a critical step is using heptane to dissolve lipids away from intact adipocytes and from adipocytes that lyse during the process. In our experience, this step substantially increases reproducibility and yield by preventing RNA and nuclei from being lost to the lipid layer. Continuous rotation of the tubes containing adipocytes undergoing fixation is equally important as it enhances the homogeneity with which the lipids are removed from the adipocytes. Instead of the 1% formaldehyde fixative buffer reported in much of the ChIP-seq literature21,22, it was found that reducing the concentration to 0.7% formaldehyde is necessary to avoid over-fixation. The chromatin shearing time was also optimized to 12 min to achieve an optimal chromatin size range (100-800 bp) for ChIP-seq.
The protocol was optimized for bulk transcriptome and epigenome studies. The current protocol still has its own limitations for single-cell transcriptome and epigenome applications. Considering the cell heterogeneity reported in the adiposity field23,24, this isolation method could be further developed to be adapted for single-cell assays. In such contexts, adipocyte-restricted markers such as boron-dipyrromethene (BODIPY)25 and perilipin-1 (PLIN1)26, ASC-127, or adipocyte-specific nuclei markers could be valuable in enabling the quantitation of additional, functional, relevant cellular parameters28. Apart from the application to genomic studies, this protocol might also improve adipocyte proteomic studies, which have an extra hurdle due to the high lipid content in adipocytes29.
This protocol can also be used to successfully extract human adipocyte nuclei (data not shown), extending its application to research on human obesity.
The authors have nothing to disclose.
We are indebted to the MPI-IE optical imaging, sequencing, bioinformatic core, and personnel. This work was supported by funding from the MPG, the Van Andel Research Institute, the European Union's Horizon 2020 research, NIH (R01HG012444 and R21HG011964), the Marie Skłodowska-Curie grant agreement No 675610, and the Federal Ministry of Education and Research under the Project Number 01KU1216 (Deutsches Epigenom Programm, DEEP).
16% Formaldehyde Solution (w/v). Methanol-free | ThermoFisher SCIENTIFIC | 28908 | |
1-bromo-3-chloropropane | SIGMA | B9673 | |
5 M NaCl | invitrogen | AM9759 | |
Automated electrophoresis instrument (2100 Bioanalyzer Instrument) | Agilent Technologies | G2939BA | |
BODIPY | ThermoFisher SCIENTIFIC | D3922 | |
Collagenase Type 2 | Worthington Biochemical Corp. | 43D14184B | |
Complete, EDTA-free, Protease inhibitor cocktail (PIC) | Roche | 4693159001 | EDTA free |
DAPI | ThermoFisher SCIENTIFIC | D1306 | |
DMEM (+ GlutaMAX) | life technologies | 61965-026 | |
DNA purification kit (PCR purification kit) | Qiagen | 28104 | |
DNA quantification instrument (Qubit 4 Fluorometer) | Fisher SCIENTIFIC | Q33238 | |
DNA quantification kit (DNA high sensitivity assay) | Agilent Technologies | 5067-4626 | |
EDTA | Fisher chimical | E478-500 | |
EGTA | Fisher chimical | O2783-100 | |
EtOH | PHARMCO | 111000200 | |
Fatty acid-free BSA | SERVA | 11932.01 | bovine albumin fraction V, fatty acid-free lyophilized |
Glycine | SIGMA | G7126-100G | |
Heptane | SIGMA | H2198-1L | |
High Sensitivity RNA Analysis | Agilent Technologies | 5067-1513 | |
Igepal | SIGMA | 18896-50ML | |
KCl | SIGMA | P3911-25G | |
Matrix filter (420um) | Tisch Scientific | ME17238 | |
Next-Generation Sequencer (HiSeq 3000 Sequencer) | Illumina | ||
Nuclease-free water | Invitrogen | 4387936 | |
PIPES | ACROS organics | 5625-37-6 | |
Proteinase K | ThermoFisher SCIENTIFIC | EO0491 | |
RNA isolation reagent (Trizol) | ThermoFisher SCIENTIFIC | 15596026 | |
RNase A, DNase-free | ThermoFisher SCIENTIFIC | EN0531 | |
Rotator (Thermo Scientific Tube Revolver Rotator) | ThermoFisher SCIENTIFIC | 88881001 | |
SDS, Sodium dodecyl sulfate | SIGMA | 8170341000 | |
Sonication tube (1 mL milliTUBE with AFA Fiber) | Covaris | 520135 | |
Sonicator (Evolution Focused-ultra-sonicator (S220 or E220)) | Covaris | 500217 or 500239 | |
Total RNA Prep kit ( Illumina Stranded Total RNA Prep kit) | Illumina | 20040525 | |
Tris-HCl | Fisher bioreagents | BP153-500 |
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