GSM9405460: WT CTL Diet S11; Mus musculus; RNA-Seq
Frozen tissues were dounce-homogenized in lysis solution - 0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM TrisCl (pH 8), 10% Triton, and 0.1 M DTT with Protector RNase Inhibitor (Roche, 03335402001). Then, nuclei isolation was performed with an iodixanol gradient. To remove the nuclear layer, 1000 μL was removed from between the 30% and 40% iodixanol layers with a P1000 and collected. 10 μL of nuclei suspension was used to count nuclei using Trypan blue. Then, the nuclei were resuspended in wash buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM TrisCl, 0.1% Tween-80), and centrifuged at 500xg for 10 min at 4ºC. Supernatant was removed, and nuclei were resuspended once more in wash buffer with 1% BSA to achieve a concentration of 1200 nuclei/μL. Approximately 20,000 nuclei/sample were used for GEM generation, barcoding, and cDNA libraries using the Chromium GEM-X Single Cell 3' Kit v4 and corresponding protocol. Pooled libraries were sequenced using the NovaSeq X Plus 25B in order to achieve >20,000 reads/nuclei.