FLASH-seq analysis of adipocyte precursor subpopulations (P1 and P2) isolated from a high-fat diet (HFD) mouse, comparing two sorting strategies: conventional manual gating and ConvexGating
FLASH-seq analysis of adipocyte precursor subpopulations (P1 and P2) isolated from a high-fat diet (HFD) mouse, comparing two sorting strategies: conventional manual gating and ConvexGating
Isolation and processing of visceral adipose tissue: The isolation of progenitor cells from the stromal vascular fraction (SVF) followed the protocol outlined by Sieckmann et al, Mol. Biol. Cell (2022). In brief, inguinal white adipose tissue (WAT) from high-fat diet-fed mice was excised, finely chopped, and enzymatically digested using 2 mg/mL collagenase II (Merck) and 15 kU/mL DNAse I (PanReac AppliChem) in PBS containing 0.5% bovine serum albumin (BSA; Sigma) at 37 °C under continuous agitation. The enzymatic reaction was terminated by adding AT buffer (PBS with 0.5% BSA). Following digestion, the cell suspension was filtered through a 100-μm strainer (Corning) and centrifuged at 500 × g for 10 min. The supernatant, which contained mature adipocytes, was removed, while the pellet comprising the stromal vascular fraction was resuspended in red blood cell lysis buffer (BioLegend) and incubated for 2 min at room temperature. This lysis step was halted by the addition of AT buffer, followed by centrifugation at 500 × g for 10 min. The isolated cells were subsequently kept in BD Omics Guard at 4°C overnight. On the next day, cells were washed, then stained with surface antibodies and incubated at 4°C for 30 minutes. Then, index sorting on a BD FACSDiscover S8 was performed.  White adipose tissue–derived adipocyte precursor subpopulations (P1 and P2) were isolated by fluorescence-activated cell sorting (FACS) into 384-well plates containing 1 µL of lysis buffer. The lysis buffer consisted of 0.2% Triton X-100, RNase inhibitor, dithiothreitol (DTT), betaine, and dNTPs. Cells were sorted directly into lysis buffer supplemented with a STRT-dT31 oligonucleotide and FLASH-UMI template switching oligo components. Plates were immediately sealed, snap-frozen on dry ice, and stored at −80°C until further processing. cDNA synthesis and amplification were performed following the FLASH-seq protocol with modifications for FLASH-UMI-seq. Reverse transcription and PCR amplification were carried out using Superscript IV reverse transcriptase with a template switching oligonucleotide containing unique molecular identifiers (UMIs) and a 5 bp spacer. RT-PCR conditions consisted of reverse transcription at 50°C for 60 minutes, initial denaturation at 98°C for 3 minutes, followed by 21 cycles of amplification (98°C for 20 s, 65°C for 20 s, and 72°C for 6 min). Amplified cDNA was purified using magnetic bead-based cleanup. Libraries were generated from normalized cDNA (~200 pg/µL) using Tn5 transposase-based tagmentation with in-house loaded Tn5 enzyme. Library enrichment PCR was performed using indexed primers compatible with Illumina sequencing. Final libraries were quantified using Qubit dsDNA HS assay and fragment size distribution was assessed using Agilent TapeStation D1000.