title: GSM4878208: LFD - Experiment 2; Mus musculus; RNA-Seq
library_construction_protocol: 10x Genomics Chromium 3' v2 Single Cell Gene Expression Kit Mice were euthanized by CO2 treatment and subsequently, subjected to cardiac perfusion using 1x PBS. eWAT was surgically removed and minced in nuclei preparation buffer (NPB) [10 mM HEPES, 1.5 mM MgCl2, 10mM KCl, 250 mM sucrose, 0.001% IGEPAL CA-630, 0.2 mM DTT, 1U/µL RNase inhibitor] on ice. The samples were further homogenized in 1.5 ml NPB using a 7 mL dounce tissue grinder, applying 10 strokes with the loose pestle and 10 strokes with the tight pestle. The homogenate was filtered through a 70 µm cell strainer and centrifuged to obtain nuclei at 1000 g for 10 min at 4 °C. The nuclear pellet was resuspended in 2 mL NPB and centrifuged at 1000 g for 10 min at 4 °C. The pellet was resuspended in nuclei resuspension buffer [20 mM Tris, 40 mM NaCl, 90 mM KCl, 2 mM EDTA, 0.5 mM EGTA, 0.001% IGEPAL CA-630, 0.2 mM DTT, 1U/µL RNase inhibitor]. The nuclei were counted using Bürker pattern counting chamber and 10,000-12,000 nuclei were submitted for snRNA-seq using the 10x Genomics system.