Figure 4.

Sorting spiral ganglion cells and RNA collection. The procedure for RNA collection is indicated. (A) Tau-EGFP SG cells were sorted with a flow cytometer. Dead cells were excluded first, which have small side scatter (SSC) and forward scatter (FSC) parameters (Left panel in A). Doublets were removed (Right panel in A). (B) EGFP positive fraction was collected as endogenous PANs, and EGFP negative fraction was collected as SGNNCs. The latter was cultured and transfected with transcription factors or control vector. (C) Tau-EGFP was upregulated in Ascl1-DsRed and NeuroD1-DsRed double transfected cells, which also expressed neuronal marker TuJ1. (D) Empty-DsRed-transfected cells expressed only DsRed, however GFP was not upregulated and TuJ1 was not expressed. (E) EGFP and DsRed double positive cells were collected as iNs from Ascl1- and NeuroD1-transfected cells. (F) DsRed single positive cells were collected as vector-control (VC) from Empty-DsRed-transfected cells. Scale bar: 50 μm.