Multi-omics analysis of eccDNA dynamics and transcriptomic changes in aged mouse lens

Description

Multi-omics profiling of eccDNA and transcriptome in aged vs. young mouse lens using Circle-seq (28), RNA-seq (16 total: 8 lens + 8 overexpression), and WGS (6). Aims to identify age-associated eccDNA signatures and functional impacts.

Steps to reproduce

Lens tissues were collected from 19‑month‑old aged ARC‑model mice and 12‑week‑old young control C57BL/6J mice. Seven mice were included in each group, and bilateral lenses were surgically dissected from euthanized animals, yielding 14 lens samples per group. Total high‑molecular‑weight genomic DNA was isolated from individual lens specimens using the MagAttract HMW DNA Kit. Trace amounts of two spike‑in plasmids (P1035 and P895) together with a 7 kb linear DNA fragment were spiked into each DNA sample to serve as external quality controls. Linear genomic DNA was selectively degraded by Plasmid‑Safe ATP‑dependent DNase (PSD) over 7 days, with ATP and PSD replenished daily. Successful removal of linear DNA and retention of circular DNA were verified by PCR assays targeting spike‑in controls and linear reference fragments. Purified eccDNA was amplified via Phi29‑mediated rolling‑circle amplification (RCA). RCA products were purified, sheared by sonication to ~400 bp fragments, and used for library construction with the MGIEasy Library Preparation Kit. Libraries were quality‑checked on a Bioanalyzer 2100 and sequenced on the MGI‑seq‑2000 platform with paired‑end 150‑bp reads. For whole‑genome sequencing (WGS), low‑input libraries were built from genomic DNA of selected remaining lens samples for ~30‑×‑depth WGS. For RNA‑seq, total RNA was extracted from lens tissues, lens‑fiber tissues, and cultured cell samples (TCMK‑1 and SRA01/04 cells transfected with synthetic eccDNA or eccRandom control). Strand‑specific mRNA libraries were prepared and subjected to next‑generation sequencing. All raw sequencing datasets generated in this study have been deposited in NCBI SRA under BioProject accession **PRJNA1513042**. Public human cataract‑lens Circle‑seq datasets (GSE207646) and human lens transcriptome datasets (GSE213546, GSE124121) were downloaded from the GEO database for secondary re‑analysis.

Institutions

• Fourth Affiliated Hospital of Harbin Medical University

  Heilongjiang, Harbin
• Harbin Medical University

  Heilongjiang, Harbin

Categories

Licence