GSM7743935: mPRAT, 2-month-old, RT, snRNA-seq; Mus musculus; RNA-Seq
Nuclei isolation from adipose tissue was performed according to a published protocol with slight modification (Van Hauwaert et al., 2021). Briefly, adipose tissues were minced in a petri dish containing 500 mL nuclei isolation buffer (NIB) that contains 250 mM sucrose (Solarbio, Cat. No. S8271), 10 mM HEPES (Solarbio, Cat. No. H8090), 1.5 mM MgCl2 (Sigma-Aldrich, Cat. No. M8266), 10 mM KCl (Sigma-Aldrich, Cat. No. P5405), 0.001% IGEPAL CA-630 (NP-40) (Sigma-Aldrich, Cat. No. I3021), 0.2 mM DTT (Sigma-Aldrich, Cat. No. D9779), and 1 U/μL RNase inhibitor (Solarbio, Cat. No. R8061) in DEPC-treated water. The samples were further homogenized using a 2 mL dounce homogenizer (Sigma-Aldrich, Cat. No. D8938), applying three strokes with the loose pestle. The homogenate was filtered through a 70 μm cell strainer (Falcon, Cat. No. 352350) and centrifuged at 500 g for 5 min at 4°C. The nuclear pellet was resuspended in 2 mL NIB and centrifuged at 300 g for 3 min at 4°C. Finally, the nuclear pellet was resuspended in 300 μL nuclei resuspension buffer that contains 2% BSA (Sigma-Aldrich, Cat. No. A1933), 1.5 mM MgCl2, 1 U/μL RNase inhibitor in PBS. All solutions were sterile filtered prior to use, and all procedures were performed on ice. Nuclei suspensions were stained with 10 μg/mL 7-AAD (Invitrogen, Cat. No. A1310) and sorted on a BD Fusion Flow Cytometer (BD FACSMelody™4-Way Cell Sorter) with a 100 µm nozzle. Nuclei were counted using a Countstar Automated Cell Counter (Countstar Rigel S5) and approximately 20,000 nuclei per sample were loaded into the 10x Chromium system using the Single-Cell 3′ Reagent Kit v3.1 according to the manufacturer's instructions (10X Genomics). GEM-Reverse Transcription was performed with a thermal cycler using the following program: 53℃ for 45 minutes, 85℃ for 5 minutes, and held at 4℃. After reverse transcription and cell barcoding, emulsions were lysed and cDNA was isolated and purified with Cleanup Mix containing DynaBeads and SPRIselect reagent (Thermo Scientific), followed by PCR amplification. The cDNA was then evaluated using an Agilent Bioanalyzer. Libraries were prepared according to the manufacturer's instructions and purified for quality assessment. snRNA-seq