100 and 40 μm cell strainers

These experiments were carried out in accordance with the protocol approved by the Committee on Animal Research at Nara Institute of Science and Technology and the RIKEN Animal Experiment Committee. Subcutaneous fat tissues from three- to four-month-old ICR male mice (n = 3 per sample, two biological replicate samples) were minced into small pieces and incubated with 0.4 U/mL collagenase NB4G (Serva) at 37 °C for 35 min in a shaking water bath. The digested solution was sequentially filtered through 100- and 40-μm cell strainers (Corning), followed by centrifugation at 250×g for 5 min to remove mature adipocytes. The pellet was treated with erythrocyte lysis buffer (BD Biosciences) and centrifuged at 180×g for 5 min. The nucleated cells were suspended in HBSS with 0.1% BSA, filtered through a 20-μm cell strainer (pluriSelect), and then kept on ice (Cell solution A). The cell aggregates that did not pass through the 20-μm strainer were further treated with Accutase (Thermo Fisher Scientific) at 37 °C for 15 min to dissociate them into single cells, centrifuged at 180×g for 5 min, and suspended in HBSS with 0.1% BSA (Cell solution B). Cell solutions A and B were mixed and again filtered through the 20-μm cell strainer, followed by centrifugation at 180×g for 5 min. The pellet was resuspended with HBSS with 0.1% BSA, stained with PI, and used for single-cell analysis.

Fresh frozen livers of Pdgfra^EGFP mice subjected to BDL or sham operation were sectioned into 100-μm-thick sections using cryostat on day 21 post-operation. The sections (20 mg) were collected into homogenisation tubes (BMS, Tokyo, Japan) containing 3 mm zirconia beads (BMS). The samples were homogenised in 1 mL of hypotonic buffer comprising 250 mM sucrose, 10 mM KCl, 5 mM MgCl₂, 10 mM Tris-HCl (pH 8.0), 25 mM HEPES, 0.1 mM dithiothreitol, 0.1% Triton X-100, 0.2 U/μL RNase inhibitor (Takara, Shiga, Japan), and protease inhibitor cocktail (Roche, Basel, Schweiz) for 10 s using a Shakeman homogeniser (BMS). The samples were incubated on ice for 15 min and homogenised again for 10 s using the homogeniser. The lysate was filtered through 100- and 40-μm cell strainers (CORNING), followed by filtration through a 5-mL filter cap FACS tube (CORNING). DAPI (1:5000; DOJINDO) was used to stain the DNA. From each population, 10,000 nuclei were collected using FACSAriaII (BD Biosciences, San Jose, CA, USA). Total RNA was extracted from the samples using the RNeasy Micro kit (Qiagen, Hilden, Germany). The cDNA was synthesised from the extracted RNA using Maxima H reverse transcriptase (Thermo Fisher Scientific).