Illumina HiSeq 4000 paired end sequencing; Single-nucleus RNA-seq of liver tissue of 3 months old mice to investigate crosstalk between ploidy and zonation
Nuclei were isolated from frozen livers from young C57BL/6 mice (3 month old) using the protocol described in Krishnaswami et al. 2016 (Nature Protocols, 11: 499-524). Nuclei were purified using Iodixanol gradient (Optiprep, D1556, Sigma Aldrich Chemie). Nuclei were stained with Hoechst and FACS sorted according to their ploidy level into tubes containing PBS/BSA 0,05%. The 4n nuclei samples were loaded in a Chromium Single Cell A Chip (10X Genomics, PN-120236) with the addition of the snRNAseq2 extra lysis reagents (NP-40 and triton-X) and were processed through the Chromium Single Cell Platform using the Chromium Single Cell 3â€² Library and Gel Bead Kit v2 (10X Genomics, PN-120237) as per manufacturers instructions. After cDNA amplification, 25% of the cDNA was processed for Library preparation (10X Genomics, PN-120237) as per the manufacturer's instructions. The final libraries were quantified and pooled for sequencing.