title: GSM8654578: mWAT tissue, CD feed 5w, HFD feed 4w; Mus musculus; RNA-Seq
library_name: GSM8654578
library_construction_protocol: Nuclei isolation of mWAT for snRNA-seq Nuclei isolation was performed as previously described (Van Hauwaert et al., 2021). Adipose tissue that was connected to the small intestine and colon was collected as mWAT. The dissected tissue was minced with scissors in 500 ml nuclei isolation buffer (NIB) that contains 250 mM sucrose (Solarbio, S8271), 10 mM HEPES (Solarbio, H8090), 1.5 mM MgCl2 (Sigma-Aldrich, M8266), 10 mM KCl (Sigma-Aldrich, P5405), 0.001% IGEPAL CA-630 (NP-40) (Sigma-Aldrich, I3021), 0.2 mM DTT (Sigma-Aldrich, D9779), and 1 U/μl RNase inhibitor (Solarbio, R8061) dissolved in DEPC-treated water (Thermo Fisher, R0601). The tissue was further homogenized with a 2 ml Dounce homogenizer (Sigma-Aldrich, D8938) for three strokes with the loose pestle, before filtering through a 70-μm cell strainer (Falcon, 352350). The nuclei pellet was obtained by centrifuging the homogenate at 500 g for 5 min at 4°C, followed by another centrifugation at 300 g for 3 min at 4°C in the NIB. The nuclei were finally resuspended in 300 μl nuclei resuspension buffer containing 2% BSA (Sigma-Aldrich, A1933), 1.5 mM MgCl2, and 1 U/μl RNase inhibitor in dissolved PBS. snRNA-seq library preparation and sequencing Nuclei suspension was stained with 7-AAD (Invitrogen, A1310) and subsequently sorted on a BD FACSMelody 4-Way Cell Sorter (BD Biosciences) with a 100-µm nozzle. Number of nuclei was assessed using a Countstar Rigel S5 Automated Cell Counter (Countstar). 20,000 nuclei per sample were loaded onto the 10x Genomics Chromium controller (10x Genomics). Libraries were prepared using the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1, 16 rxns (10x Genomics, PN-1000121). Libraries were sequenced on an Illumina NovaSeq 6000 sequencer with 150 bp paired-end reads, aiming for a coverage of approximately 50,000 raw reads per nucleus.