Illumina NovaSeq 6000 paired end sequencing; scATAC-seq of mouse liver tissue derived from young and old mice
scATAC-seq of mouse liver tissue derived from young and old mice
Liver nuclei (n=4) were prepared from frozen tissue specimens by crushing and dounce homogenising the tissue in 1 ml EZbuffer (SIGMA) (20 strokes with loose and a tight pestle, respectively) and spun 5 min at 300 g. The pellet was incubated on ice for 20 min in EZ-buffer supplemented with DNAseI NEB M0303S (4 units/ml) and 1X DNAseI buffer. Equal volume of EZ-buffer was added and samples were spun 5 min at 500 g and incubated again 10 min on ice in EZ-buffer supplemented with DNAseI NEB M0303S (8 units/ml) and 1X DNAseI buffer. Equal volume of EZ-buffer was added, and samples were spun 5min at 500g, resuspended in NSB (1087.5 µl 1XPBS, 5.5µl 2% BSA, 1.5 µl RNase Inhibitor) and filtered 3 times through a 0.22 µm strainer. For scATAC-seq, 100,000 nuclei were resuspended in 50 µl tagmentation mix. scATAC-seq targeting 4000 cells per sample was performed using a beta version of Chromium Single Cell ATAC Library and Gel Bead kit (10x Genomics, 1000110) according to the manufacturer's instructions.