GSM6697839: EODF; Mus musculus; RNA-Seq
Inguinal WAT (inWAT) from each treatment were dissected from mice (6 mice for each treatment) and cut into small piece (1 mm3) with scissors. Quickly transfer inWAT into a pre-wat 100 μm cell strainer and all together in a petri dish on ice. Add 3 ml ice-cold nuclei preparation buffer [(NPB) 10 mM HEPES (15630-080, Life Technologies) (pH 7.5), 1.5 mM MgCl2 (M1028, Sigma-Aldrich), 10 mM KCl (P9541, Sigma-Aldrich), 250 mM sucrose (S7903, Sigma-Aldrich) and 0.1% IGEPAL CA-630 (NP-40) (I8896, Sigma-Aldrich) in DEPC water] to the inWAT pieces and use syringe plunger to mince inWAT with strainer. Be aware that inWAT should be bathed with ice-cold NPB. Minced inWAT will eventually flow through the strainer and stay at petri dish. Wash the plunger and strainer with 2 ml NPB then filter through the strainer. The homogenate (now is at petri dish, inWAT debris is at upper side of the strainer) was then transferred to a new, pre-cooled 50 ml tube on ice. Centrifuge at 1,000 g for 10 minutes at 4℃. After aspirate the lipid layer, nuclear pellet was resuspended in 100 μl ice-cold nuclei resuspension buffer [(NRB) 1%BSA, 2 mM MgCl2 (M1028, Sigma-Aldrich) and 0.04 U RNase inhibitor (03335399001, Roche) in 1X PBS]. The nuclei were counted using Countess® II FL Automated Cell Counter (Thermo Fisher Scientific) and 12,000 nuclei were submitted for snRNA-seq using the 10X Genomics system. 10X Genomics sequencing libraries were acquired by 10X Chromium Single Cell mRNA Seq 3' v3.1 reagent kit. Nuclei solutions containing 700-1200 nuclei/μl were loaded into Chromium Controller. snRNA-seq