Protocol for isolating small cytosolic dsDNA from cultured murine cells

Zhizheng Dai; Hongyu Ji; Anqi Zheng; Ai-Long Huang; Kai-Fu Tang

doi:10.1016/j.xpro.2024.102998

Protocol for isolating small cytosolic dsDNA from cultured murine cells

STAR Protoc. 2024 Apr 3;5(2):102998. doi: 10.1016/j.xpro.2024.102998. Online ahead of print.

Authors

Zhizheng Dai¹, Hongyu Ji¹, Anqi Zheng², Ai-Long Huang³, Kai-Fu Tang⁴

Affiliations

¹ Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing 400016, P.R. China.
² Key Laboratory of Diagnosis and Treatment of Severe Hepato-Pancreatic Diseases of Zhejiang Province, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325015, P.R. China.
³ Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing 400016, P.R. China. Electronic address: ahuang@cqmu.edu.cn.
⁴ Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing 400016, P.R. China. Electronic address: tangkaifu@cqmu.edu.cn.

Abstract

We recently identified a class of small cytosolic double-stranded DNA (scDNA) approximately 20-40 bp in size in human and mouse cells. Here, we present a protocol for scDNA isolation from cultured murine cells. We describe steps for cytosolic compartment separation, DNA isolation in the cytosolic fraction using phenol-chloroform extraction, and ethanol precipitation. We then detail procedures for denaturing purified cytosolic DNA through urea polyacrylamide gel electrophoresis and obtaining scDNA in the cytosolic DNA fraction via gel purification. For complete details on the use and execution of this protocol, please refer to Liu et al.¹.

Keywords: Cell culture; Cell separation/fractionation; Molecular Biology.