FACS-Sorted Hair Cells - RNAseq

Cochleas and utricles were dissected from mice expressing EGFP under the Pou4f3 promoter, specific to hair cells. Cells were dissociated and sorted by FACS; hair cells (GFP+) and surrounding cells (GFP-) were separately collected for RNA extraction. Poly-A mRNA was selected, and the 3' end ~500bp of each transcript was amplified and reversely transcribed to cDNA, and the cDNA was sequenced on the Illumina next-generation sequencing platform. This was done at four developmental stages (E16, P0, P4, and P7) for both cochlea and utricle. The raw reads were quality filtered and mapped against the NCBI build 37/mm9 mouse genome assembly on the cloud server at DNAnexus using the 3SEQ workflow. The read counts of all RefSeq genes were summarized, normalized, and statistical tests of differential gene expression for each gene under various comparison schemes were performed using the DESeq package in R/BioConductor.

Publication to cite:
Scheffer DI, Shen J, Corey DP, Chen ZY. (2015) Gene expression by mouse inner ear hair cells during development. J Neurosci. 35:6366-80.

Funding: NIDCD R01DC002281 to DPC; NIDCD R03DC013866 and HHF Emerging Research Grant to JS; NIDCD R01DC006908 to ZYC; Howard Hughes Medical Institute

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Adult Cochlear Inner and Outer Hair Cells - GeneChip

Inner hair cells (IHCs) and outer hair cells (OHCs) were isolated from P25 to P30 adult mice (CBA/j) cochleae. Two thousand IHCs and 2,000 OHCs were individually collected using the suction pipette technique. Total RNA was extracted using the Qiagen RNeasy Mini Plus Kit. Separate IHC and OHC RNAs were split into three for separate technical replicates. Gene expression profiles were determined by GeneChip Mouse Gene 2.0 ST Arrays. All the CEL files from the Mouse Gene 2.0 ST arrays were normalized using Expression console (Affymetrix, Santa Clare, CA, USA). These data were exported into CLC Main Workbench (Cambridge, MA, USA) to perform analysis and statistics. Gene expression values from each experiment for either type of hair cells as well as means and standard deviations from three technical replicates are all included in the searchable dataset. Additional 2,000 OHCs were collected for verification (data are presented as OHC4 in the downloadable Excel file).

Publication to cite:
Liu HZ, Pecka J, Zhang Q, Soukup GA, Beisel KW, He DZ (2014). Characterization of transcriptomes of cochlear inner and outer hair cells. J Neurosci. 34:11085-11095.

Funding: R01 DC004696 to DZH.

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Otic Progenitor Cells - RNAseq and ChIPseq

An immortalized multipotent otic progenitor (iMOP) cell line was generated by transient expression of c-Myc in Sox2-expressing otic progenitor cells. The procedure activated endogenous c-Myc expression in the cells and amplified existing Sox2-dependent transcripts to promote self-renewal. Downregulation of c-Myc expression following growth factor withdrawal resulted in a molecular switch from self-renewal to otic differentiation. Mouse cochlear progenitor cells were dissected at E12-E14 and transduced with a c-Myc retrovirus to create clonal cell lines that were passaged up to 100 times in culture. These lines maintained their multipotent cell identity, expressing early markers of the cochlear proneurosensory region. In vitro, the cells divided in response to bFGF but entered into quiescence upon removal of growth factor and express the cell cycle control genes Rb and p27. When cultured under the appropriate conditions, progenitor cells can differentiate into neurons, hair cells and supporting cells.

Total RNA was extracted from progenitor cells cultured in bFGF, EGF or without growth factors. Ribosomal RNA was removed and mRNA was reversely transcribed to cDNA via random priming. ChIP-Seq was accomplished by immunoprecipitating endogenous RNA PolII by RNA polymerase II clone 4H8 (Abcam Cat#ab5408), c-Myc by c-Myc N-262 (Santa Cruz Cat# sc-764), and Sox2 by Sox2 clone 6F1.2 EMD (Millipore Cat# 17-657). Libraries were generated from the cDNA or ChIP DNA, and sequenced on the Illumina next-generation sequencing platform. RNAseq analysis was performed as mentioned above and ChIPseq analysis was done using the MACS algorithm.

Publication to cite:
Kwan KY, Shen J, Corey DP. C-MYC transcriptionally amplifies SOX2 target genes to regulate self-renewal in multipotent otic progenitor cells. Stem Cell Reports. 2015 Jan 13;4(1):47-60. doi: 10.1016/j.stemcr.2014.11.001.

Funding: NIDCD R01DC002281 to DPC; NIDCD R03DC013866 and HHF Emerging Research Grant to JS; AHRF grant to KYK; Howard Hughes Medical Institute

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Stereocilia Proteomics - Mass Spectrometry

Stereocilia from chick utricle were purified using the agarose twist-off method, and constituent proteins identified by mass spectrometry. Isolated proteins were digested with trypsin; tryptic peptides were separated by reverse-phase liquid chromatography, then analyzed with an ion-trap mass spectrometer (Thermo Orbitrap Elite) in the MS/MS mode. Spectra were searched against the Ensembl version 66 database using the Andromeda search engine (Cox et al., 2011); proteins were identified and quantified the iBAQ algorithm (Schwanhäusser et al., 2011) using MaxQuant (Cox & Mann, 2008).


Cox J & Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol. 26, 1367-1372.

Cox J et al. (2011) Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res. 10, 1794-1805.

Schwanhäusser B et al. (2011) Global quantification of mammalian gene expression control. Nature 473, 337-342.

Publication to cite:
Shin JB, Krey JF, Hassan A, Metlagel Z, Tauscher AN, Pagana JM, Sherman NE, Jeffery ED, Spinelli KJ, Zhao H, Wilmarth PA, Choi D, David LL, Auer M, Barr-Gillespie PG. (2013). Molecular architecture of the chick vestibular hair bundle. Nature Neuroscience. 2013 Mar;16(3):365-74. doi: 10.1038/nn.3312.

Funding: NIDCD R01DC002368 and R01DC011034 to PGG.

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Auditory and Vestibular Ganglion Neurons - GeneChip

Spiral and vestibular ganglion neurons were collected at six developmental stages (E12, E13, E16, P0, P6 and P15). E12 and E13 spiral and vestibular ganglia, as well as E16, P0, P6, and P15 vestibular ganglia, were microdissected from embryos or pups generated by CD1 x Rnx-cre; Z/EG crosses. E16, P0, P6, and P15 spiral ganglia were dissected from embryos or pups generated by CD1 x MafB-GFP+/- crosses, and GFP+ spiral ganglion neurons were dissociated, then sorted and collected by FACS. RNA was isolated from samples and linearly amplified, then hybridized to Affymetrix Mouse 430 v.2 GeneChips.

Publication to cite:
Lu CC, Appler JM, Houseman EA, Goodrich LV. (2011) Developmental profiling of spiral ganglion neurons reveals insights into auditory circuit assembly. J Neurosci. 31:10903-18.

Funding: NIDCD R01-009223 to LVG; March of Dimes Grant 1-FY08-381 to LVG; NOHR PKG Foundation Grant in Auditory Science to CL; NIH/NIDCD Grant F32 DC008228 to CL

Download XLS ↓ | Download rawdata (GSE29766)