Hepatocyte polyploidization during ageing protects against transcriptional dysregulation and chronic liver disease
Nuclei were isolated from frozen livers, harvested from young (3 months) and old (24 months) C57Bl/6J, Hnf4a+/0, Cebpa+/0 and Ctcf+/0 mice. Methodology to isolate and purified nuclei is described in Strzelecki et al. 2022 (JoVE, 190). Briefly, iodixanol gradient (optiprep, D1556, Sigma Aldrich Chemie) was used to purify nuclei. All nuclei were stained with Hoechst and FACS sorted according to their ploidy level into tubes containing PBS/BSA 0.05%. cDNA were made on 384-well plates using the single -nucleus-RNA-seq2 protocol based on a modified version of SMART-Seq Single Cell kit (Takara). ERCC spike-in RNA (Ambion) (0.25 μL diluted at 1:400.000) was added to the RT-Mix (5μL final reaction). After cDNA amplification, 25% of the cDNA was processed for library preparation using the standard Illumina Nextera XT. DNA Sample Preparation kit (Illumina, #FC-131-1096) and the combination of 384 Combinatorial Dual Indexes (Illumina- Set A to D, #FC-131-2001 to FC-131-2004). Final libraries were quantified and pooled for sequencing.