Single-cell analysis RNA sequencing of whole mouse skin at embryonic day 13.5 and 14.5

A major challenge in delineating molecular and cellular events that precede appendage morphogenesis stems from the inability to distinguish quantitative molecular differences between cells that lack histological distinction. The hair follicle (HF) dermal condensate (DC) is a cluster of cells critical for HF development and regeneration. Events that presage emergence of this distinctive population are poorly understood. Using unbiased single-cell RNA sequencing and in vivo methods, we infer a sequence of transcriptional states through which DC cells pass that begins prior to HF morphogenesis. Our data indicate that Wnt/b-catenin signaling is required to progress into an intermediate stage that precedes quiescence and differentiation. Further, we provide evidence that quiescent DC cells are recent selective progeny of proliferating cells present prior to morphogenesis and that are identified in the peri-DC zone during DC expansion. Together, these findings provide an inferred path of molecular states that lead to DC cell differentiation. Overall design: 6 embryo samples total, 3 collected at E13.5 and 3 collected at E14.5. Embryonic dorsolateral/flank skin was micro-dissected and pooled (3-4 embryos per sample) and dissociated into a single-cell suspension using 0.25% trypsin (Gibco, Life Technologies) for 30 minutes at 37° C. Single-cell suspensions were then stained with DAPI (Fisher Scientific, NBP2-31156) just prior to fluorescence-activated cell sorting. DAPI-excluded live skin cells were sorted on a BD Facs Aria II (Biosciences) sorter with a 100 µm nozzle. Cells were sorted in bulk for 10X Genomics platform. Chromium Single cell 3' Library and Gel Bead Kit v2 Chromium Single Cell 3' Library & Gel Bead Kit v2 (PN- 120237), Chromium Single Cell 3' Chip kit v2 (PN-120236) and Chromium i7 Multiplex Kit (PN-120262) were used according to the manufacturer's instructions in the Chromium Single Cell 3' Reagents Kits V2 User Guide. After cDNA libraries were created, they were subjected to HiSeq 2500 (Illumina) sequencing.