characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-05-08T00:14:13Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-05-08T00:14:13Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA112993471
characteristics.characteristic: External Id
characteristics.text: Copenhagen University, BRIC
characteristics.characteristic: INSDC center name
characteristics.text: 2023-05-08T00:14:13Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-05-08T00:14:13Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS14988495
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12898:Sample 343
characteristics.characteristic: Submitter Id
characteristics.text: 45
characteristics.unit: year
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: human
characteristics.characteristic: common name
characteristics.text: Protocols: Cells were plated in technical triplicates in 24w plates and collected either at t0 (upon expansion, without differentiation) at t14 (after adipogenic differentiation: see treatment protocol). At the collection timepoints, cell culture medium was discarded and 300 ul of TRIzol (Sigma, #T3934) was applied directly on the plate. The cell lysate was immediately transferred to RNAse-free tubes and stored at -80º until RNA extraction. 2-3 cm3 biopsies from Subcutaneous (SC), Omentum (OM), Perirenal (PR) and Mesocolic (MC) adipose tissues were washed in PBS to remove excess blood, weighted and finely minced using scissors. Minced adipose tissue was incubated with 0.28 U/ml of liberase TM (Roche #05401119001) in DPBS with calcium and magnesium (Gibco #14040091) for 60 min at 37 °C under agitation. Vigorous shaking was performed after 45 min of incubation to increase the yield of recovered SVF cells. The digested tissue was mixed with an equal volume of 1% human albumin (CSL Behring) in DPBS −/− (Gibco #14190094) to stop the lysis. Following a 5-min centrifugation at 400 g at room temperature, floating lipids and mature adipocytes were discarded by aspiration and the resuspended SVF pellet was sequentially filtered through 100-μm and 40-μm cell strainers to ensure a single cell preparation. To lyse red blood cells, pelleted SVF was resuspended in VersaLyse solution (Beckman Coulter #A09777) according to the manufacturer’s recommendations and washed once with 1% albumin solution. Obtained red blood cell-free SVF suspension was then plated for experiments at a density of at least 100’000 cells per square centimeter in high glucose MEMalpha GlutaMax medium (Gibco #32561037) supplemented with 5% human platelet lysate (Sigma #SCM152) and 50 μg/ml Primocin (InvivoGen #ant-pm-2). Adipogenic differentiation: after cells were confluent for at least 24h, they were treated with induction cocktail (high glucose DMEM (#61965), 10% FBS, 50 μg/ml Primocin, 0.5 mM IBMX (Sigma #15879), 1 μM dexamethasone (Sigma #D2915), 1.7 μM insulin (Sigma #19278), 0.2 mM indomethacin (Sigma #I7378) for 7 days, followed by a maintenance cocktail (high glucose DMEM, 10% FBS, 50 μg/ml Primocin, 1.7 μM insulin) for another 7 days. No medium refreshment was performed between these two timepoints. RNA extraction was performed on two different days, one for each experimental batch. The Zymo kit for 96-RNA extractions (Zymo, #R2054) was used according to the manufacturer's protocol. 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 second-strand synthesis via the nick translation method using 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 were 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) using the following 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 a 0.6x ratio to remove the fragments < 300 nt. The resulting libraries were profiled using a High Sensitivity NGS Fragment Analysis Kit (Advanced Analytical, #DNF-474) and measured using a Qubit dsDNA HS Assay Kit (Invitrogen, #Q32851) prior to pooling and sequencing.
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: normal
characteristics.characteristic: disease
characteristics.text: D28
characteristics.characteristic: individual
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: perirenal fat
characteristics.characteristic: organism part
characteristics.text: 2
characteristics.characteristic: replicate
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: female
characteristics.characteristic: sex
characteristics.text: Sample 343
characteristics.characteristic: title