title: GSM6604432: E15.5 embryo, Ebf2-GFP enriched; Mus musculus; RNA-Seq
library_construction_protocol: Embryos were collected at the specified timepoints and head and limbs were removed. The dorsal upper back portion was minced and digested with either 0.25% trypsin (Fisher) or Collagenase D: 6.1 mg ml−1 (Roche) and Dispase II: 2.4 mg ml−1 (Roche). Samples were placed at 37 °C with constant agitation (200 r.p.m), followed by quenching for 30 min with an equal volume of complete medium (DMEM/10% FBS). Dissociated cells were suspended using a P1000 pipette and filtered through a 100-μm filter. Cells were then pelleted at 400g for 4 min, and red blood cells were lysed in 155 mM NH4Cl, 12 mM NaHCO3 and 0.1 mM EDTA for 4 min. An equal volume of complete medium was added, and the cells were filtered through a 40-μm filter for downstream analyses. Isolated cells were suspended in FACS buffer (HBSS, 3% 0.45 µM filtered FBS; Thermo Fisher). Cells were stained in FACS buffer with antibodies for 1 h at 4 °C in the dark. Cells were washed twice with cold FACS buffer and sorted on a FACS Aria cell sorter (BD Biosciences) with a 100-μm nozzle. All compensation was performed at the time of acquisition in BD FACS DIVA software (8.0.1) using single stained cells and compensation beads. Single cells were flow sorted to isolate live (DAPI(−)) cells and remove debris. We enriched for mesenchymal, DM and brown adipose lineage cells as follows.  (i) E11.5 and E12.5 DM and mesenchymal cells based on cell surface expression of PDGFRa and/or LRIG1 [30% PDGFRa+, LRIG1(-) cells; 25% PDGFRa(-), LRIG1+ cells, 30% PDGFRa+, LRIG1+ cells, 15% PDGFRa(-), LRIG1(-) cells]. (ii) E13.5-E15.5 fibroblasts through selection of PDGFRa+ cells [75% PDGFRa+; 25% PDGFRa(-)]. (iii) E10.5, E14.5, and E15.5 brown adipose lineage cells based on expression of Early B Cell Factor-2 (Ebf2)-GFP [75% Ebf2-GFP+; 25% Ebf2-GFP(-)]. Single cell libraries were prepared  according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics) and sequenced on an Illumina HiSeq 4000 or an Illumina NovaSeq 6000 by the University of Pennsylvania Next Gen Sequencing Core or CHOP Center for Applied Genomics Sequencing Core. Reads were aligned using Cell Ranger software from 10x Genomics.