GSM8502271: AL, ATAC, islets, rep1; Mus musculus; OTHER
Single nuclei were isolated from snap-frozen isolated islet pellets from AL, CR, and HFD mice after 2 months on diet. Nuclei were isolated using the 10x Genomics protocol for nuclei isolation from embryonic mouse brain. Briefly, the islet pellet was lysed and homogenized in 0.1x Lysis Buffer solution (Tris-HCl 10 mM, NaCl 10 mM, MgCl2 3 mM, Tween-20 0.01%, IGEPAL CA-630 0.01%, Digitonin 0.01%, BSA 1%, DTT 1 mM, RNase inhibitor 1 U/ul). Then, the lysate was incubated on ice for 5 min, pipette mixed and incubated on ice for additional 10 min. Next, chilled wash buffer (Tris-HCl 10 mM, NaCl 10 mM, MgCl2 3 mM, Tween-20 0.1%, BSA 1%, DTT 1 mM, RNase inhibitor 1 U/ul) was added to the sample, pipette mixed, and the sample was filtered through a Flowmi Cell Strainer. Then, the pellet was washed with chilled wash buffer and the nuclei concentration was deter-mined using Trypan Blue Stain (0.4%) and hemocytometer. The final nuclei concentration (1000 nuclei/ul) was resuspended in chilled Diluted Nuclei Buffer (10x Genomics). Isolated nuclei were processed for transposition, barcoding, library construction and sequencing according to 10x Genomics Guidelines for Single Cell Multiome ATAC + Gene Expression.