title: NextSeq 500 sequencing; 3'RNA-seq of murine ASPCs derived from P0, P16, 4wo, 7wo and 11wo-mice
library_construction_protocol: Isolation of SVF cells from newborn (P0) mice Burgeoning depots of inguinal subcutaneous adipose tissue were dissected from new-born (P0) wild-type C57BL/6J mice and transferred into PBS. The tissues were then transferred into collagenase solution (see above) and incubated for 1 h at 37 °C under agitation (130 rpm on a vertical shaker), after which they were resuspended after 45 min of digestion. The cell suspension was then filtered through a 100-µm, then 40-μm cell strainer in order to generate a single cell preparation. Next, the cells were pelleted by a 5-min centrifugation at 400 g at room temperature and a red blood cell lysis was performed by incubating the pellet in a homemade red blood cell lysis buffer (see above) for 5 min, followed by two washes (a 5-min centrifugation at 400 g, room temperature) with a homemade FACS buffer (see above).  Isolation of the mouse stromal vascular fraction (SVF) Subcutaneous inguinal adipose tissue depots were dissected from wild-type C57BL/6J male and female mice of different ages (P16 (16 days post-birth), 4-week-old, 7-week-old, 11-week-old) 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 (BO).  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− CD45− TER119–) population was selected, followed by a Lin−SCA1+ selection (ASPCs), and finally, ASPC cells that were negative or positive for CD142 marker: Lin− SCA1+ CD142− (CD142− ASPCs) and Lin− SCA1+ CD142+ (CD142+ ASPCs, Aregs).  Wild-type C57BL/6J male and female adult mice were euthanised at CO2. Subcutaneous inguinal adipose tissue depots were dissected from wild-type C57BL/6J male and female adult mice and transferred into ice cold PBS for further processing. Total, CD142− and CD142+ ASPCs (Aregs) freshly isolated form the indicated age groups (P0, P16, 4wo, 7wo and 11wo) were collected into Tri-Reagent (Molecular Research Center #TR118). Direct-zol RNA kit (Zymo Research #R2052) was used to extract RNA according to the manufacture's specifications. 3'end bulk mRNA sequencing was performed following the BRB-seq strategy as previously described (Alpern et al., 2019). In brief, 7-200 ng of total RNA from each sample was reverse transcribed in a 96-well plate using SuperScriptTM II Reverse Transcriptase (Lifetech 18064014) with individual barcoded oligo-dT primers, featuring a 12-nt-long sample barcode (IDT). Double-stranded cDNA was generated by the second strand synthesis via the nick translation method. For that, a mix containing 2 μl of RNAse H (NEB, #M0297S), 1 μl of E. coli DNA ligase (NEB, #M0205 L), 5 μl of E. coli DNA Polymerase (NEB, #M0209 L), 1 μl of dNTP (10 mM), 10 μl of 5x Second Strand Buffer (100 mM Tris, pH 6.9, (AppliChem, #A3452), 25 mM MgCl2 (Sigma, #M2670), 450 mM KCl (AppliChem, #A2939), 0.8 mM β-NAD (Sigma, N1511), 60 mM (NH4)2SO4 (Fisher Scientific Acros, #AC20587), and 11 μl of water was added to 20 μl of ExoI-treated first-strand reaction on ice. The reaction was incubated at 16 °C for 2.5 h. Full-length double-stranded cDNA was purified with 30 μl (0.6x) of AMPure XP magnetic beads (Beckman Coulter, #A63881) and eluted in 20 μl of water.  The Illumina-compatible libraries were prepared by tagmentation of 10-40 ng of full-length double-stranded cDNA with 1 µl of in-house produced Tn5 enzyme (11 μM). After tagmentation, the libraries where purified with DNA Clean and Concentrator kit (Zymo Research #D4014), eluted in 20 µl of water and PCR amplified using 25 μl NEB Next High-Fidelity 2x PCR Master Mix (NEB, #M0541 L), 2.5 μl of each i5 and i7 Illumina index adapter (IDT) following this program: incubation 72 °C—3 min, denaturation 98 °C—30 s; 15 cycles: 98 °C—10 s, 63 °C—30 s, 72 °C—30 s; final elongation at 72 °C—5 min. The libraries were purified twice with AMPure beads (Beckman Coulter, #A63881) at 0.6x ratio to remove the fragments < 300 nt. The resulting libraries were profiled with a High Sensitivity NGS Fragment Analysis Kit (Advanced Analytical, #DNF-474) and measured with the Qubit dsDNA HS Assay Kit (Invitrogen, #Q32851) prior to pooling and sequencing using the Illumina NextSeq 500 (Illumina).