DNBSEQ-G400 paired end sequencing; scRNA-seq of mouse liver tissue derived from young and old mice
scRNA-seq of mouse liver tissue derived from young and old mice
Livers were dissociated using the Miltenyi liver perfusion kit (130-128-030) following the manufacturer's instructions. For the sc-RNA sequencing experiment the isolated hepatocytes were washed two times with staining buffer (1x PBS, 2mM EDTA, 0.5%BSA) and 1 million hepatocytes were stained with Hoechst (15μg/ml) and Reserpine (5uM) for 30min at 37°C. Dead cells were excluded with PI staining (1μg/ml). Cells were sorted using a BD FACSARIA IIIU or Fusion Cytometer and 130um nozzle. The data were analysed using the BD FACSDiva and FlowJo softwares. part of nucleic acid library construction protocol scRNA-seq libraries were generated using an early version of the Smart-seq3xpress protocol. Reaction conditions were identical to the published final protocol deposited on protocols.io (https://www.protocols.io/view/smart-seq3xpress-yxmvmk1yng3p/v2). Briefly, cells were sorted into 300 nl of lysis buffer containing 0.5 uM oligo-dT primer overlayed with 3 ul Silicon Oil (100 cSt; Sigma-Aldrich). Next, reverse transcription was carried out in 400 nl using 0.75 uM Smart-seq3 TSO. Preamplification of cDNA was performed in 1 ul using SeqAmp polymerase (Takara Bio) for 15 cycles. Subsequently, amplified cDNA was diluted with 9 ul H2O and we used 1 ul for downstream tagmentation using 0.002 ul TDE1 enzyme per cell. Final, indexed libraries were pooled per 384-well plate, cleaned and equimolarly pooled before sequencing.