GSM8279032: WAT_NC; Mus musculus; RNA-Seq
Brown and white adipose tissues were isolated from chow diet-fed Glsfl/fl mice and GlsAdipoq-Cre mice (n=5 for each genotype) and flash frozen in liquid nitrogen. Samples from each depot and genotype were pooled for the nuclei isolation. Briefly, frozen samples were minced into 1-2 mm2 pieces and homogenized in 3 mL cold lysis buffer (5 mM CaCl2, 3 mM magnesium acetate (Mg(C2H3O2)2), 2 mM EDTA, 0.5 mM EGTA, 10 mM TrisHCl, 1 mM DTT [all Sigma], 1X protease inhibitor [11836170001, Roche] and 0.2 U/µL RNase inhibitor [10777019, Invitrogen] in nuclease free water) using a gentleMACS Dissociator (Miltenyi Biotec). Subsequently, 3 mL lysis buffer with 0.4% Triton X-100 (X100, Sigma-Aldrich) was added and the tubes were centrifuged briefly. The lysates were filtered through a 70 µm cell strainer (BD Biosciences), the filters were washed with 2 mL lysis buffer and centrifuged at 1000 x g for 5 minutes at 4 °C using a swinging bucket rotor. The pellet was resuspended into 1 mL nuclei buffer (2% BSA in PBS supplemented with 0.2U/µl RNase inhibitor and 1 mM DTT) and filtered through a 35 µm cell strainer. Nuclei were stained with 10 µg/mL DAPI (D9542, Sigma-Aldrich), and positive nuclei were sorted into Eppendorf tubes coated with nuclei buffer. The tubes were centrifuged at 1000 x g for 5 minutes at 4 °C and resuspended into nuclei buffer for counting. Nuclei suspensions were diluted to a concentration of 1100 nuclei/µL and loaded onto a 10X Chip G (10X Genomics). Chromium Single-Cell v3.1 kit was used to prepare libraries according to the manufacturer's protocol. The libraries were sequenced on a Nextseq 2000 platform (Illumina).