Single cell RNA sequencing of the steroidogenic lineage in the male adrenal cortex

To achieve single-cell characterization of the adrenal cortex transcriptome, we selectively labeled the steroidogenic adrenocortical cells using a transgenic Cre-LoxP approach. We combined Sf1-Crehigh mice with Rosa26mTmG animals to obtain mice expressing fluorescent reporter proteins at the cell membrane (mTmG) of cells that express or have expressed the transcription factor SF-1 (Steroidogenic Factor 1, also AdBP4, encoded by NR5A1). We prepared a single-cell suspension by combining mechanic and enzymatic dissociation at low temperatures to preserve the viability of steroid cells and their transcriptional states. Fluorescence-activated cell sorting (FACS) was then used to isolate GFP-positive cells representing the steroidogenic lineage. GFP-positive cells include the progenitor populations as well as differentiated cells (zona glomerulosa and zona fasciculata cells). Overall design: A total of 26 male mouse adrenals (15 week-old) was harvested and dissociated into single cell suspension. Steroidogenic cells were isolated by FACS and analyzed by single cell RNAsequencing.