Published: March 17th, 2023
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.
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.
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
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 chromatin accessibility of pure adipocyte populations isolated from different fat depots.
We observed that the mCherry-labeled ....
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 is also important to prevent adipocyte nucleus damage during nucleus isolation and sorting. The samples must be handled gently via low-speed ....
Copyright © 2023 MyJoVE Corporation. All rights reserved