C1 single cell auto prep ifc for preamp

For single-cell qPCR, we captured single cells of P5 endolymphatic sacs using medium-sized microfluidic chips (C1 Single-Cell Auto Prep IFC for PreAmp) as outlined in the Fluidigm protocol (PN 100–6117 G1). Mixes for lysis, RT, and specific target amplification were prepared from the Single Cell-to-Ct qRT-PCR kit (Ambion, Austin, TX) and pre-designed TaqMan gene expression assays (Thermo Fisher Sientific). In addition to canonical cell markers, putative MRC and RRC markers at P5 (Figure 2—source data 3) were selected for analysis in an arbitrary manner.
After 18 cycles of preamplification, expression levels were measured by qPCR on a 96.96 Dynamic Array IFC using the Fluidigm BioMark HD system. cDNA from single cells was selected for qPCR in the same way as it was selected for RNA-seq. The threshold of cycles (C_t) values were calculated with Fluidigm Real-time PCR analysis software (RRID:SCR_15686) with the following settings: quality threshold of 0.65; a linear (derivative) baseline correction; and auto (detectors) method. We defined gene expression levels as log₂ expression = LOD – C_t, in which we set C_t = 28 as LOD. We used the log₂ expression dataset for hierarchical clustering.