Mus musculus
Protocols: Subcutaneous and visceral adipose tissue depots were dissected from male and female 8-12-week-old Pref1-RFP mice into ice cold PBS. The tissue was finely minced using scissors, transferred into collagenase (Sigma-Aldrich #C6885-1G, 2 mg/ml of collagenase buffer (25 mM NaHCO3, 12 mM KH2PO4, 1.2 m MgSO4, 4.8 mM KCl, 120 mM NaCl, 1.4 mM CaCl2, 5 mM Glucose, 2.5% BSA, pH = 7.4)) and incubated for 1 hour at 37_C under agitation. Following a 1 min centrifugation at 400g, at room temperature (RT), floating mature lipid-filled adipocytes were aspirated and the suspension was filtered through a 40 µm cell strainer to ensure a single cell preparation. Next, a red blood cell (RBC) lysis was performed by incubating the pelleted cells in the RBC lysis buffer (154 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) for 5 min, followed by two washes (5 min centrifugations at 400g, RT) with FACS buffer (PBS with 3% foetal bovine serum (FBS) (Gibco #10270-106), 1mM EDTA, 1 % penicillin/streptavidin (P/S) (Gibco #15140122)). The isolated single cell suspension was diluted to 0.75 or 1 * 107 cells/ml with FACS buffer (PBS with 3% FBS, 1mM EDTA, 1% P/S and the following fluorophore-conjugated antibodies were added (in titration-determined quantities): anti-mouse CD31-AF488, anti-mouse CD45-AF488, anti-mouse TER119-AF488 (all BioLegend #303110, #304017 and #116215 respectively) for selecting the Lin- population, anti-mouse SCA1-PE-Cy7 (BioLegend #122513), anti-mouse CD34-BV421 (BioLegend #119321) and anti-mouse CD29- PerCP-efluor710 (eBiosciences #46-0291) to enrich the Lin- population with ASPCs. 1,100 cells (550 RFP+ and 550 RFP-) at the concentration of 200 cells/µl were sorted by FACS directly into the C1 Single-Cell Auto Prep Array IFC 10-17µm chip (Fluidigm #100-5760) for 96 cells with the efficiency of ~10%. The presence of the cells was validated by microscopy visualisation (Olympus Cell Xcellence, objective: UPLFLN 10x/0.30). anti-mouse CD142-PE (SinoBiological #50413-R001) and anti-ABCG1 antibody (Invitrogen #PA5-13462) conjugated with APC for separating negative and positive populations for the given marker. The cells were incubated with the cocktail of antibodies on ice for 20 min protected from light, after which they were washed and stained with DAPI (Sigma #D9542) or propidium iodide (PI) (Molecular Probes #P3566) for assessing viability and subjected to FACS using a Becton Dickinson FACSAria II sorter. Compensation measurements were performed for single stains using compensation beads (eBiosciences #01-2222-42). The same number of cells was sorted directly into flat bottom microscopy-adapted cell culture plates (Corning #353219), cultured to confluence in high glucose DMEM medium (Gibco #61965026) supplemented with 10% FBS and 1% P/S, and treated with a single dose of white adipocyte differentiation induction cocktail (0.5 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma #15879), 1 µM dexamethasone (Sigma #D2915), 1 µg/ml insulin (Sigma #19278), followed by maintenance treatment (1 µg/ml insulin) every 48h. The culture was carried out until day 10-12 post-induction, when cells were stained for imaging or harvested for RNA extraction. RNA isolation of sorted cells: Live cells were collected in RLT+ lysis buffer (Qiagen #1053393) and flash-frozen on dry ice. Cell lysates were homogenized with QIAshredders prior to RNA isolation using the RNeasy Plus Micro Kit (Qiagen #74034). Reverse transcription was performed using the QuantiTect whole transcriptome kit (Qiagen #207045) following manufacturerês recommendations for a standard-yield reaction (2 h of amplification time) or SuperScriptÂ VILO cDNA Synthesis Kit (Invitrogen #11754050). RNA isolation of differentiated cells: mRNA was isolated and transcribed into cDNA using the MultiMACS cDNA Synthesis Kit (Miltenyi #130-094-410) or collected into Tri-Reagent (Molecular Research Center #TR118). Direct-zol RNA kit (Zymo Research #R2052) was used to extract RNA, followed by reverse transcription using SuperScriptÂ VILO cDNA Synthesis Kit (Invitrogen). Bulk mRNA sequencing was performed in a barcoded fashion, using a time, labor and cost-efficient preparation of a single Nextera-compatible cDNA library containing up to 96 (and potentially more) different RNA samples (Alpern et al., in preparation). The protocol is adopted from SCRB-seq, developed for single cell transcription profiling, with several important modifications. For the current series of experiments, 50 ng of total RNA from each sample was reverse transcribed in a 96-well plate using Maxima H Minus RT (Thermo) with individual oligo-dT primers, featuring a 6 nt long multiplexing barcode, and template switch oligo (Microsynth). Specifically, each oligo-dT primer is biotinylated and has the following structure: 5ê- ACACTCTTTCCCTACACGACGCTCTTCCGATCT[BC6] [N15] [T30] VN-3ê, where, [BC6] = 6 nt barcode specific to each well; N15 stretch of random nucleotides forming a Unique Molecular Identifier (UMI). Thus, for each well, we have generated a unique combination of barcodes and UMIs to identify each well (sample) and transcript. Next, all the samples were pooled together, purified using the DNA Clean and Concentration kit (Zymo), and treated with exonuclease I (NEB). The full-length cDNA library was amplified using a single primer and purified with AMPure beads (Beckman Coulter). The sequencing library was prepared by tagmentation of 10 ng full length cDNA with an in house made Tn5 transposase at 55çC for 9 minutes. Tagmented DNA was purified with the DNA Clean and Concentration kit and PCR amplified using NEBNext High-Fidelity 2X PCR Master Mix (NEB) with an i7 adapter identical to Illumina Nextera and custom i5 (Microsynth). The PCR reaction was then purified twice with AMPure beads and the average fragment size of the library was evaluated using a Fragment Analyzer (Advanced Analytical) prior to paired-end sequencing with NextSeq 500 (Illumina).
ENA first public
2017-11-30
ENA last update
2017-06-05
External Id
SAMEA104112713
INSDC center alias
EPFL SV IBI-SV UPDEPLA
INSDC center name
EPFL SV IBI-SV UPDEPLA
INSDC first public
2017-11-30T17:01:52Z
INSDC last update
2017-06-05T11:47:21Z
INSDC status
public
Submitter Id
E-MTAB-5787:Sample 19
age
8 to 10
broker name
ArrayExpress
cell type
mesenchymal stem cell of adipose
common name
house mouse
genotype
Pref1-RFP
growth condition
induction of adipocyte differentiation
individual
pool 4
organism part
adipose tissue
phenotype
CD142 negative
sample name
E-MTAB-5787:Sample 19
strain
C57BL/6