Extended Data Fig. 6: FACS purification of and RNA extraction from OHCs from an E18.5 Insm1^GFP.Cre/+ embryo.

a–c, Forward and side light scattering were used to exclude dead cells and debris (a) and aggregates (≥2 cells) (b, c). d, Live cells were gated in green and red (PerCP-Cy5, to assess autofluorescence) channels to define the GFP⁺ (green dots) and GFP^– (red dots) sorting windows. e, Myosin VIIa immunoreactivity and DAPI stain of cells collected through cytospinning after FACS confirm that most of the 547 sorted GFP⁺ cells are hair cells. This verification was done on all hair cell pools sorted (three pools per genotype). Inset is a representative merged image of one sorted OHC at high magnification. In this pool, no autofluorescent cells were collected. f, RT–qPCR after cell sorting (mean ± s.e.m.) reveals that, compared with GFP^– cells, GFP⁺ cells express the hair cell marker gene Myo7a and not the supporting cell marker genes S100 and Hes5. g, To ensure the quality of the extracted RNA, the RIN score was determined using a BioAnalyzer. g, Similar RIN scores were obtained from all pools of OHCs examined (including the three per genotype used for RNA-seq in Fig. 4a).