Illumina HiSeq 4000 paired end sequencing; Single-nucleus RNA-seq2 of liver tissue of 3 months old mice to investigate crosstalk between ploidy and zonation
Single-nucleus RNA-seq2 of liver tissue of 3 months old mice to investigate crosstalk between ploidy and zonation
Nuclei was 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 was purified using Iodixanol gradient (Optiprep, D1556, Sigma Aldrich Chemie). Nuclei were stained with Hoechst and FACS sorted according to their ploidy level diploid (2n) and tetraploid (4n) in 384-well plates. cDNA were made on 384-well plates using single-nucleus-RNA-seq2 protocol based on a modified version of SMART-Seq v4 Ultra Low input RNA (Takara). ERCC spike-in RNA (Ambion) (0.25 µL diluted at 1:100,000 and 1:300,000) was added to the RT-Mix (5µL final reaction). Sequencing libraries were prepared using Illumina Nextera XT 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) following the manufacturer instructions but miniaturizing the reactions 10x using a liquid handling robot (Mosquito HV).
Experimental Factor: single cell identifier
do25573
Experimental Factor: ploidy
2n