Illumina HiSeq 2500 paired end sequencing; Single-cell profiling of developing mouse adipocyte progenitor cells
Single-cell profiling of developing mouse adipocyte progenitor cells
Epididymal fat pads from 4 adult male Wt1-GFP mice (3-4 months old) were dissected and made into a single cell suspension.  SVF (stromal vascular fraction) was suspended in PBS (5% FCS) and filtered (40 uM) before staining with surface antibodies for FACS isolation. The Lineage panel of antibodies was purchased from BD (559971), CD31 (eBioscience, 46-0311-80, 1:100), IgM (eBioscience, 25-5790-81, 1:100), and CD34 (BioLegend, 119311, 1:50). For multicolour analysis and sorting, colour compensation samples were produced by singly staining SVF cells with one antibody of each of the combined fluorochromes as well as by FMO (Fluorochrome Minus One) controls. Single cell FACS sorting in 384 well plates was performed using a FACS Flow Cytometer (BD Aria II). Single adipocyte precursor cells were sorted in 384 well plates, directly into lysis buffer containing ERCC92 RNA mixture 1 at a dilution of 1:10^6 (ThermoFisher).  Single cell cDNA was generated by the SMARTSeq2 protocol.  Multiplexed single-cell libraries were prepared for high throughput sequencing using the Nextera XT protocol (Illumina).