title: NextSeq 500 sequencing; 3'RNA-seq of murine post-differentiation CD142− ASPCs treated with retinoic acid (RA)
design: 3'RNA-seq of murine post-differentiation CD142− ASPCs treated with retinoic acid (RA)
library_construction_protocol: Isolation of the mouse stromal vascular fraction (SVF) Subcutaneous inguinal adipose tissue depots were dissected from wild-type C57BL/6J male and female adult mice and transferred into PBS. The tissues were subsequently minced using scissors, transferred into collagenase (Sigma-Aldrich #C6885-1G, 2 mg/ml of homemade collagenase buffer (25 mM NaHCO3, 12 mM KH2PO4, 1.2 m MgSO4, 4.8 mM KCl, 120 mM NaCl, 1.4 M CaCl2, 5 mM Glucose, 2.5% BSA, pH = 7.4)) and incubated for 65-70 min at 37 °C under agitation (130 rpm on a vertical shaker), after which they were resuspended after 45-50 min of digestion. The cell suspension was then diluted with PBS (with the aim of diluting the collagenase and stopping/slowing down the digestion), and filtered through a 100-µm, then 40-μm cell strainer in order to generate a single cell suspension. Next, the cells were pelleted by a 5 min centrifugation at 400 g at room temperature and red blood cells were lysed by incubating the pellet with a homemade red blood cell lysis buffer (154 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) for a maximum of 5 min, followed by two washes (a 5 min centrifugation at 400 g, room temperature) with a homemade FACS buffer (PBS with 3% foetal bovine serum (FBS) (Gibco #10270-106), 1 mM EDTA, 1% penicillin–streptomycin (Gibco #15140122)).  FACS-based cell isolation The SVF cell suspension was diluted to 1 × 10^7 cells/ml with a homemade FACS buffer (see above) and the following fluorophore-conjugated antibodies were added (in titration-determined quantities, Suppl. Table 3): anti-mouse CD31–AF488, anti-mouse CD45–AF488, anti-mouse TER119–AF488 (BioLegend #303110, #304017 and #116215, respectively) to select the Lin− population; anti-mouse SCA1-PE-Cy7 (BioLegend #122513) to enrich the Lin− population with ASPCs, and anti-mouse CD142-PE (# ORB507485)  The cells were incubated with the antibodies on ice for at least 30 min and protected from light, after which they were washed with the FACS buffer, stained with propidium iodide (Molecular Probes #P3566) for assessing viability, filtered through a 40-µm cell strainer and subjected to FACS with the use of a Becton Dickinson FACSAria II sorter. Compensation measurements were performed for single stains using positive/negative compensation beads (eBiosciences #01-2222-42).  The following gating strategy was applied while sorting the cells: first, the cells were selected based on their size and granularity/complexity (side and forward scatter), followed by a double elimination of any events that could represent more than one cell. Next, the Lin−(CD31−