laser capture micro dissection combined with next generation sequencing analysis of cell type specific deafness gene expression in the mouse cochlea

Accepted Manuscript Laser-capture micro dissection combined with next-generation sequencing analysis of cell type-specific deafness gene expression in the mouse cochlea Shin-ya Nishio, Yutaka Takumi, Shin-ichi Usami PII: S0378-5955(15)30050-2 DOI: 10.1016/j.heares.2017.02.017 Reference: HEARES 7334 To appear in: Hearing Research Received Date: 11 July 2015 Revised Date: 25 December 2016 Accepted Date: 28 February 2017 Please cite this article as: Nishio, S.-y., Takumi, Y., Usami, S.-i., Laser-capture micro dissection combined with next-generation sequencing analysis of cell type-specific deafness gene expression in the mouse cochlea, Hearing Research (2017), doi: 10.1016/j.heares.2017.02.017 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain ACCEPTED MANUSCRIPT Hearing Research Laser-capture micro dissection combined with next-generation sequencing analysis Department of Otorhinolaryngology, Shinshu University School of Medicine M AN U SC Shin-ya Nishio1,2 , Yutaka Takumi1, Shin-ichi Usami1,2 RI PT of cell type-specific deafness gene expression in the mouse cochlea 3-1-1 Asahi, Matsumoto 390-8621, Japan Department of Hearing Implant Sciences, Shinshu University School of Medicine TE D 3-1-1 Asahi, Matsumoto 390-8621, Japan Correspondence to Shin-ichi Usami: EP Department of Otorhinolaryngology Shinshu University School of Medicine AC C 3-1-1, Asahi, Matsumoto 390-8621, Japan Tel: +81-263-37-2666 Fax: +81-263-36-9164 E-mail:usami@shinshu-u.ac.jp ACCEPTED MANUSCRIPT Abstract Hereditary hearing loss is one of the most common congenital sensory disorders worldwide, with approximately one hundred genes estimated to be involved Recent RI PT advances in molecular genetic analysis technology using next-generation sequencing have accelerated the exploration of novel genes involved in genetic deafness, and also allowed the identification of mutations in each patient in a relatively short period SC Cochlear implantation (CI), which directly stimulates the cochlear nerves, is the most effective and widely used medical intervention for patients with severe to profound M AN U sensorineural hearing loss The outcomes of CI, however, vary among patients The etiology of the hearing loss is speculated to have a major influence of CI outcomes, particularly in cases resulting from mutations in genes preferentially expressed in the neurons of the spiral ganglion region TE D To elucidate more precise gene expression levels in each part of the cochlea, we performed laser-capture micro dissection in combination with next generation-sequencing analysis and determined the expression levels of all known EP deafness-associated genes in the organ of Corti, spiral ganglion, lateral wall, and spiral limbs The results were generally consistent with previous reports based on AC C immunocytochemistry or in situ hybridization As a notable result, the genes associated with many kinds of syndromic hearing loss (such as Clpp, Hars2, Hsd17b4, Lars2 for Perrault syndrome, Polr1c and Polr1d for Treacher Collins syndrome, Ndp for Norrie Disease, Kal for Kallmann syndrome, Edn3 and Snai2 for Waardenburg Syndrome, Col4a3 for Alport syndrome, Sema3e for CHARGE syndrome, Col9a1 for Sticker syndrome, Cdh23, Cib2, Clrn1, Pcdh15, Ush1c, Ush2a, and Whrn for Usher syndrome and Wfs1 for wolfram syndrome) showed higher levels of expression in the spiral ACCEPTED MANUSCRIPT ganglion than in other parts of the cochlea This dataset will provide a base for more detailed analysis in order to clarify gene AC C EP TE D M AN U SC RI PT functions in the cochlea as well as predict CI outcomes based on gene expression data ACCEPTED MANUSCRIPT Introduction Hearing loss is one of the most common congenital or early onset sensory disorders, RI PT appearing in one out of 700 to 1,000 newborns, with 50%–70% of cases attributable to genetic causes (Morton and Nance, 2006) Inherited hearing loss demonstrates great heterogeneity and approximately one hundred genes are estimated to be involved SC (Hereditary hearing loss homepage; http://hereditaryhearingloss.org/) Regarding the medical treatment of hearing loss, cochlear implantation (CI), which M AN U directly stimulates the cochlear nerves, is the most effective and widely used medical intervention for patients with severe to profound sensorineural hearing loss However, the outcomes of CI vary among patients Many factors, including medical, educational, and environmental, are presumed to affect CI performance With regard to the medical TE D factors affecting CI outcomes, diagnostic age, pre-operative hearing thresholds, duration of pre-operative hearing loss, implantation timing, associated mental retardation or other complex symptoms have been reported (Artières, et al., 2009, Kasai, et al., 2012, et al., 2015) EP Forli, et al., 2011, Iwasaki et al., 2012, Beadle, et al., 2005, Calmels, et al., 2004 Birman, AC C Recent advances in genetic analysis technology using Massively Parallel DNA Sequencing have accelerated the exploration of novel genes involved in deafness (Walsh, et al., 2010, Rehman, et al., 2010, Brownstein, et al., 2011, Schraders, et al., 2011, Azaiez, et al., 2015) and also have made possible the comprehensive genetic testing of all known deafness causing genes in a shorter period (Shearer, et al., 2010, Lin et al., 2012, Yan, et al., 2013, Mutai et al., 2013, Nishio, et al., 2015a, 2015b) As a result, it is now possible to identify the etiology of hearing loss in each patient prior to ACCEPTED MANUSCRIPT cochlear implantation This information should be considered as an additional medical factor affecting CI outcome, because differences in the etiology of hearing loss will reflect different hearing loss mechanisms RI PT Recently, Eppsteiner et al hypothesized that CI outcomes in patients with mutations in genes preferentially expressed in the spiral ganglion neurons might worse than those of patients with an intra-cochlear etiology (Eppsteiner, et al., 2012) They also proposed SC some a number of genes typically associated with poor CI performance (CHD7, DDP1/TMM8 alpha) and variable CI performance (MYH9, TMPRSS3, POU3F4) M AN U (Eppsteiner, et al., 2012) This hypothesis appears quite reasonable; however, our results for four TMPRSS3 cases treated with electric acoustic stimulation (EAS) revealed relatively good performance in comparison with patients with hearing loss of other etiologies (Miyagawa, et al., 2013, 2015) TE D To elucidate more the precise gene expression levels in the spiral ganglions and other parts of the each cochlea parts, we performed laser-capture micro dissection in combination with next-generation sequencing (LMD-NGS) analysis and identified the EP expression levels of all known deafness-causing genes in each part of the cochlea As the laser captured parts, we selected four cochlear regions in (the organ of Corti, spiral AC C ganglion, lateral wall, and spiral limbs) as these regions were representative of four cochlear functions (mechano-electrical transduction, production of endolymph, production of the tectorial membrane, and signal transmission for the central nerve system) and have clear landmarks that make them easily distinguishable from other parts of cochlea This dataset will provide a base for more detailed analysis in order to predict CI outcomes on the basis of gene expression data ACCEPTED MANUSCRIPT Material and Methods Tissue dissection and RNA extraction RI PT Eight Four male C57BL/6J mice aged 12 weeks (SLC, Shizuoka, Japan) were euthanized by decapitation under deep anesthesia with an intraperitoneal injection containing 75 mg/kg Ketamine (Daiichi Sankyo, Tokyo, Japan) and 32.4 mg/kg SC Pentobarbital Sodium (Kyoritsu, Tokyo, Japan) Inner ears were rapidly extracted from the temporal bone and transferred into RNAlater solution (Ambion, Life Technologies M AN U Co., Grand Island, NY) After removing the otic capsule, the cochlea was dissected All of these dissections were performed in RNAlater solution to prevent RNA degradation After dissection, the cochlea samples were immersed in 10% sucrose followed by 30% sucrose in 0.1M phosphate buffer (pH7.4) for hours each and then embedded into TE D OCT compound (Sakura Finetek, Tokyo, Japan) for sectioning at 14 µm using a Leica CM1900 cryostat (Leica Mannheim, Germany) Cryosections were fixed on Membrane Slide NF 1.0 PEN (Carl Zeiss, Munchen, Germany) and stained with a 1% cresyl violet EP acetate (SIGMA, St Louis, MO, USA) ethanol solution After washing with 100% AC C ethanol on ice and drying, laser-capture micro dissection was performed using a PALM laser-capture micro dissection system (Carl Zeiss) according to the manufacture’s protocol Eight Four micro sections of each of the organ of Corti (including the basement membranes), lateral wall, spiral ganglion, and spiral limbus (mainly the interdental cell region) were captured, respectively Micro dissectioning of each part of the cochlea was performed four times twice using samples from different animals so that a total of 16 samples were prepared Total RNA samples were extracted using a QIAGEN RNeasy Micro Kit (QIAGEN, ACCEPTED MANUSCRIPT Hilden, Germany) according to the “PALM protocols for RNA handling” and “PALM protocols for RNA extraction from frozen sections” (Carl Zeiss) RI PT Amplicon Library Preparation Prior to amplicon library preparation, reverse transcription was performed using a High Capacity RNA-to-cDNA Kit (Applied Biosystems, Life Technologies, Foster City, CA, SC USA) according to the manufacturer’s procedure with some modifications (input RNA sample quantities were too small for standard measurement procedures such as Qubit, M AN U BioAnalyser or UV spectrometer, so an equal volume of total RNA samples extracted from laser-capture micro dissections were used as templates for reverse transcription.) Custom amplicon primers for the 123 124 mouse target genes were prepared as an Ion AmpliSeq™ RNA Custom Panel (Applied Biosystems, Life Technologies) Amplicon TE D libraries for next-generation sequencing analysis were prepared with a custom designed panel, Ion AmpliSeq™ Library Kit 2.0 (Applied Biosystems, Life Technologies), and Ion Xpress™ Barcode Adapter 1-16 Kit (Applied Biosystems, Life Technologies) EP according to the manufacturers’ instructions After library preparation, equal amount of AC C libraries for samples were pooled into one tube Emulsion PCR and Sequencing The emulsion PCR was performed with the Ion OneTouch™ System and Ion P1™ Template OT2 200 Kit v3 (Applied Biosystems, Life Technologies) according to the manufacturer’s instructions After the emulsion PCR, template-positive Ion Sphere™ Particles were enriched with Dynabeads® MyOne™ Streptavidin C1 Beads (Applied Biosystems, Life Technologies) and washed with Ion OneTouch™ Wash Solution ACCEPTED MANUSCRIPT included in the kit This process were performed using an Ion OneTouch™ ES system (Applied Biosystems, Life Technologies) After the Ion Sphere Particle preparation, next-generation sequencing was performed with an Ion Proton™ system using the Ion Technologies) according to the manufacturer’s instructions SC Base Call and Data Analysis RI PT P1™ 200 Sequencing Kit v3 and Ion P1™ Chip (Applied Biosystems, Life The sequence data were processed with standard Ion Torrent Suite™ Software ver 4.2 M AN U and mapped to the mouse cDNA sequence for 123 124 target genes in multi-FASTA format with a Torrent Mapping Alignment Program After the sequence mapping, the mapped read number for each gene was calculated using coverage analysis plug-in software included in the Torrent Suite™ Software The total mapped read number for TE D each sample was converted to 1M reads and the relative value of the expression level for each gene was calculated (RPM: reads per million reads) EP Microarray analysis To obtain a whole cochlear gene expression profile, we performed cDNA microarray AC C analysis Total RNA samples from the whole cochlea of C57BL6/J mice (12 weeks old) were prepared as described elsewhere with some modifications (in our previous report, we divided cochlea into pieces whereas we did not divide the cochlea in this study) (Yoshimura, et al., 2014) After RNA sample preparation, total RNA (25ng each) was reverse-transcribed using a Low Input Quick Amp Whole Transcriptome Labeling Kit (Agilent Technologies, Santa Clara, CA) and labeled with T7 RNA polymerase mix and cyanine 3-CTP according to the manufacturer’s instructions Each cochlea sample ACCEPTED MANUSCRIPT was hybridized to SurePrint G3 Mouse GE 8x 60K Microarrays (Agilent Technologies), which were spotted with 60K probes (4 microarrays were used for each sample) Fluorescence intensities were measured with an Agilent Microarray Scanner (Agilent) RI PT using scanning protocols specific for each microarray assay The expression data were extracted from raw microarray image files using Agilent Feature Extraction Image Analysis Software (Version 10.7.3.1) After quantification, mean fluorescence intensities M AN U whole cochlea gene expression level control SC of microarray samples for 123 124 deafness causing genes were calculated as the Quantitative RT-PCR To confirm the next-generation sequencing analysis results, qPCR was performed on deafness genes and house-keeping control genes The TaqMan® probe for each gene TE D was selected from the TaqMan® Gene Expression Assay system (https://products.appliedbiosystems.com/ab/en/US/adirect/ab?;cmd=ABGEK eywordSearch, Applied Biosystems) Gapdh and Actb were chosen as internal control EP genes The estimated gene expression level (EL) was normalized to the internal control AC C gene expression level and data are presented as the mean of log2EL Ethical Statement All experimental procedures were performed in accordance with the regulations for animal experimentation of Shinshu University These experiments were approved by the Shinshu University Institutional Animal Care and Use committee ACCEPTED MANUSCRIPT Figure Gene expression levels of each cochlear part analyzed by quantitative RT-PCR Expression values of each gene are indicated as the relative value to the Actb expression RI PT level The gene expression level of each gene measured by quantitative RT-PCR was AC C EP TE D M AN U SC generally comparable to that of the LMD-NGS analysis ND: not detected ACCEPTED MANUSCRIPT RI PT Table Gene expression levels profiles analyzed by LMD-NGS analysis for the organ of Corti, lateral wall, spiral limbus and spiral ganglion in the mouse cochlea Fluorescence intensities from the cDNA microarray analysis of the whole mouse cochlea samples are also shown as SC a control Actg1 DFNA20/26 Adcy1 Atp6v1b1 DFNB44 Renal tubular acidosis with deafness Bdp1 DFNB49 Bsnd Bartter syndrome, type 4a Cabp2 DFNB93 Ccdc50 DFNA44 Cdh23 DFNB12/USH1D Ceacam16 AD-NSHL Cemip AD-NSHL Chd7 CHARGE syndrome Cldn14 Cib2 DFNB29 DFNB48/USH1J Clic5 DFNB102 Lateral Wall Spiral Limbus 54,886 75,616 73,652 85,142 80,665 41 185 946 1,836 1,194 563* 3,885 16 53 74 33 159 711 81 293 25* 222 776 104 50 41 265 763 105 534 7,741 243 500 43 45 32 93 19 218,148 46* 157,920 583* 117,268 5,988 11,179 9,827 164* N/A 305 97 34* 178 3207 13,735 76 6,403 174 4,593 1,845 12,308 64,235 456 16,583 72,081 108 69 156 1,373 24 2,968 AC C Spiral Ganglion cDNA micro array (FU) Organ of Corti TE D Locus/Disease EP Gene Name M AN U Relative gene expression levels based on LMD-NGS analysis (reads / 1M reads) Whole cochlea ACCEPTED MANUSCRIPT 27 64 1,420 1,577 USH3 197 33 39 3,821 147 Coch DFNA9 57,176* 442,265** 398,098 7,470* 95,453 Col11a1 STL2 2,482 11,227 6,965 3,207 645 Col11a2 DFNA13/DFNB53/STL3 1,343* 5,939 5,932 1,449* 4,333 Col2a1 STL1 2,441 2,457 792 1,518 6,359 Col4a3 Alport syndrome 126 552 357 870 27 Col4a4 Alport syndrome 21,623 10,220 4,973 18,506 463 Col4a5 Alport syndrome 25,291 Col4a6 X linked-NSHL 59 Col9a1 STL4 43 Col9a2 STL5 4,268 Col9a3 AR-NSHL 141 Crym AD-NSHL Dfna5 DFNA5 Dfnb59 DFNB59 Diaph1 DFNA1 Dspp DFNA39 Edn3 WS4 Ednrb WS4 Elmod3 DFNB88 Eps8 AR-NSHL Ercc3 XPB Esrrb DFNB35 SC Clrn1 RI PT 57 M AN U PRLTS3 2,841 5,190 629 451 103 22* 173 253 41 20* 110 24 8,999 5,091 76* 13,784 88 54* 427 348 1,403 1,383 448 1,414 827 16 17 11 66 896 8,228 14 10 109 314 934 7,222 3,394 17,160 600 923 469 339 949 13 213 496 88* 565 14 1,077 374 401 1,170 915 84 84 2,816 156 5,481 51 120 42 139 485 108 217 797 304 994 73,145 49,242 26,060 100,824 16,733 AC C EP TE D Clpp ACCEPTED MANUSCRIPT 475 218 433 11,624 89,093 69 9,546 24,829 990 Jackson-Weiss syndrome 147 22 Foxi1 PDS 216 76 Gipc3 DFNB15/72/95 32 38 Gjb2 DFNA3A/DFNB1A 17,475 20,811 Gjb3 DFNA2B/DFNB91 1,705 909 Gjb6 DFNA3B/DFNB1B 1,653 34,144 Gpsm2 DFNB82 1,857 Grxcr1 DFNB25 23 Grxcr2 DFNB101 35 Hars2 PRLTS2 2,041 Hgf DFNB39 291 Hsd17b4 PRLTS1 Ids Mucopolysaccharidosis II Idua Mucopolysaccharidosis Ih/s Ildr1 DFNB42 Kal Kallmann syndrome Kars Kcnj10 DFNB89 Enlarged vestibular aqueduct, digenic Kcnq4 DFNA2A Lars2 PRLTS4 Lhfpl5 DFNB66/67 Loxhd1 DFNB77 17 49 393 53 134 64 11 22 13 10,369 103* 78,727 555 3,484 11 6,882 1,714 92,545 SC Fgfr2 M AN U DFNA10 36 28* 85 494 11 10 47 20 34 87 149 5,287 1,416 35,856 1,766 116 62 204 169 38 2,772 1,972 28,064 8,109 18,149 349 2,230 387 1,677 169 91 19,873 3,644 12,141 763 1,569 16,364 311 916 569 668 80* 2,242 N/A 13,493 10,337 2,409 13,769 9,127 63 54 3,163 254 26,623 404 1,555 765 368 21,36 1,253 57 448 10,776 1,363 7,213 98 45 131 711 362 360 115* 892 169 TE D Eya4 RI PT 181 EP BOR1 AC C Eya1 ACCEPTED MANUSCRIPT DFNB63 33 19 10 1,656 579 Marveld2 DFNB49 98 164 33 1,997 179 Mitf WS2A 183 843 Msrb3 DFNB74 921 Myh14 DFNA4 Myh9 RI PT lrrc51 564 6,654 3,951 333* 8,123 315 10,391 5,191 5,178 610* 52 DFNA17 123 6,106 9,714 125 11,205 Myo15 DFNB3 51 19 351 Myo1a DFNA48 250 186 106 223 125 Myo3a DFNB30 112 Myo6 DFNA22/DFNB37 Myo7a DFNA11/DFNB2/USH1B Ndp Norrie Disease Otoa DFNB22 Otof DFNB9 Otog AR-NSHL Oyogl DFNB84 P2rx2 DFNA41 Pax3 WS1/WS3 Pcdh15 DFNB23/USH1F Pdzd7 USH2C Pnpt1 DFNB70 Polr1c TCS3 Polr1d TCS2 Pou3f4 DFNX2 M AN U 515 67* 330 18 7,348 3,486 2,527 327 164 59 130 560 2,610 787 4,886 1,535 15,038 46,471 30,663 533 140 516 30* 211 2,325 11,408 25 5,694 140 671 61 109 35 181 N/A 149 869 10,959 313 3,992 470 367 105* 1,351 108 101 70 43 211 68 36 39 14 55 32 21 23 9* 59 657 6,942 26,792 6,871 69,880 3,221 77 57 99 245 4,109 26 27,389 2,582 77 122 1,583 68 1,543 AC C EP TE D 37,528 (DFN3) SC 59 ACCEPTED MANUSCRIPT 36 42 67 117 10,569 13,445 4,558 25,573 4,569 DFNB84 68 89 Rdx DFNB24 887 4,106 Sall1 Townes-Brocks syndrome 36,815 798 Sans USH1G 237 408 Sema3e CHARGE syndrome 74 2,978 Serpinb6b AR-NSHL 1,142 4,849 Six1 DFNA23/ BOS3 10,139 1,665 Six5 BOS2 Slc17a8 DFNA25 Slc26a4 DFNB4/PDS 346* Slc26a5 DFNB61 2,990 Smpx DFNX4 Snai2 WS2D Sox10 WS4 Strc DFNB16 Syne4 DFNB76 Tbc1d24 DFNA65/DFNB86 Tcof1 TCS1 Tecta DFNA8/12/DFNB21 Tjp2 DFNA51 Tmc1 DFNA36/DFNB7/11 Tmie DFNB6 Tmprss3 DFNB8/ 10 48 229 108 540 62,096 3,531 557 2,130 292 102* 556 590 36 15,246 1,108 1,157 25,058 6,585 5,138 33,851 8,026 2,351 4,953 136 1,152 27,980 17,456 52,737 489 40,450 39,878** 2,628* 47,435 989* 721* 4,748 54 23,094 20,919 1,536* 40,701 529 202 82 40 1,216 411 5,937 36 4,981 71 11,416 51 102 33* 277 176 384 341 119 15,868 N/A 230 434 154 13,501 108 71 83 41 107 1,856 13 2* 50 11 6,707 4,770 792 20,560 8,419 55 324 38* 883 431 275 1,479 164 725 368 18,067 9,323 15,296 170* 1,953 27 63,162 (DFN6) SC Ptprq (DFN2) M AN U DFNX1 TE D Prps1 RI PT 54 EP DFNA15 AC C Pou4f3 ACCEPTED MANUSCRIPT DFNA56 107 58 32 128 13 Tprn DFNB79 8,321 34,197 6,083 75,563 1,084 Triobp DFNB28 156 182 Tspear DFNB98 370 Ush1c DFNB18/USH1C Ush2a Vlgr1 USH2A USH2C Wfs1 DFNA6/14/38 Whrn DFNB31/USH2D RI PT Tnc 226 5,735 396 190* 956 N/A 1,093 604 904 3,036 2,516 330 29 442 480 106* 1,122** 34 13 58 8,299 4,424 3,259 15,954 4,494 175 1,s851 M AN U 83 SC 22 138 30* USH: Usher syndrome, NSHL: Non-syndromic hearing loss, PRLTS: Perrault syndrome, STL: Sticker syndrome, WS: Waardenburg intensities unit TE D Syndrome, BOS: Branchiootorenal syndrome, TCS: Treacher Collins syndrome, XPB: Xeraderma pigmentosum B, FU: fluorescence * indicates statistically low gene expression compared with the averaged gene expression level of the other parts of the cochlea Table S1 AC C EP ** indicates statistically high gene expression compare with the averaged gene expression level of the other parts of the cochlea Next-generation sequencing metrics of the LMD-NGS analysis in this study RI PT ACCEPTED MANUSCRIPT Table S2 AC C EP TE D M AN U SC Espn, Kcne1, and Kcnq1 gene expression levels ACCEPTED MANUSCRIPT A E F I J LW OC D SL C OC SC H K M AN U SL L M N O EP LW TE D SL Q R AC C LW T RI PT SL B U SG P S V ACCEPTED MANUSCRIPT 3.5 2.5 1.5 0.5 ND Gapdh ND ND ND Slc26a4 Organ of Corti Lateral Wall Clrn1 Spiral Limbus Spiral Ganglion AC C EP TE D M AN U Actb expression Tmprss3 SC RI PT Reads number / 1M reads 1000000 100000 ACCEPTED MANUSCRIPT 10000 1000 100 10 Coch Ceacam16 Actg1 Esrrb Slc17a8 Otoa Tprn Eya4 Polr1c Sall1 Kars Cldn14 Smpx Slc26a4 Clic5 Rdx Cib2 Col4a4 Wfs1 Pou3f4 RI PT 100000 10000 1000 100 10 Prps1 Six1 Gjb2 Hars2 Gjb6 Tmprss3 Diaph1 Cemip Col11a1 Idua Myh14 Ids Msrb3 Lars2 P2rx2 Clrn1 Kal SC Reads number / 1M reads 1000000 100000 10000 1000 100 10 10000 1000 10 EP 100 1000000 Reads number / 1M reads Col9a2 Tjp2 Serpinb6 Sema3e Otog Syne4 Myh9 Myo6 Col11a2 Tbc1d24 TE D 100000 Ildr1 Hsd17b4 Sox10 Ndp Slc26a5 Ccdc50 Atp6v1b1 Dfnb59 Lhfpl5 Six5 Col2A1 Gjb3 Ush1c Crym Ednrb Dspp Tmie Pax3 Marveld2 Gpsm2 Ush2a Tspear Col4a3 Loxhd1 AC C Reads number / 1M reads 1000000 M AN U Reads number / 1M reads 1000000 Col4a5 100000 10000 1000 100 10 Average Kcnj10 Organ of Corti Elmod3 Kcnq4 Lateral Wall Spiral Limbus Spiral Ganglion ACCEPTED MANUSCRIPT 100000 10000 1000 100 10 Lrtomt Mitf Clpp Snai2 Ercc3 Edn3 Eya1 Sans Tmc1 Bsnd Chd7 Triobp Vlgr1 Polr1d Foxi1 Strc Cabp2 Ptprq Tnc Bdp Tcof1 Fgfr2 Col9a1 Cdh23 100000 10000 1000 10 1000000 100000 10000 1000 100 Organ of Corti Lateral Wall Spiral Limbus EP Average Pou4f3 TE D 10 AC C Reads number / 1M reads SC 100 Myo3a Otof Myo1a Col9a3 Hgf Whrn Pcdh15 Myo7a Otogl Col4a6 Esp8 Dfna5 Gipc3 Grxcr1 Myo15a Tecta M AN U Reads number / 1M reads 1000000 Adcy RI PT Reads number / 1M reads 1000000 Grxcr2 Pdzd7 Spiral Ganglion Pnpt1 ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT Determined the gene expression levels of all known deafness-associated genes in the cochlea ACCEPTED MANUSCRIPT Sample Name Total Reads Bases Over QV20 bases 1,764,914 85,692,385 79,722,502 Lateral Wall 1,211,356 51,211,173 47,180,011 Spiral Limbs 3,485,346 249,542,358 230,769,666 Spiral Ganglion 2,101,071 25,492,617 19,818,054 Organ of Corti 1,564,741 60,911,301 56,584,477 Lateral Wall 1,856,477 106,160,087 98,547,626 Spiral Limbs 1,296,536 63,063,449 58,516,209 Spiral Ganglion 1,963,105 74,090,573 68,586,630 Organ of Corti 6,523,902 617,950,327 571,341,144 Lateral Wall 3,124,391 255,286,950 236,054,026 Spiral Limbs 21,995,598 1,969,507,556 1,815,468,253 Spiral Ganglion 2,523,486 216,595,459 201,468,147 Organ of Corti 3,111,852 299,722,896 Lateral Wall 16,135,833 1,497,755,501 Spiral Limbs 14,327,511 1,412,812,737 Spiral Ganglion 3,496,710 337,377,488 311,631,935 Average 5,405,177 457,698,304 423,217,110 SC 278,180,195 1,379,650,501 1,317,954,378 M AN U TE D EP AC C RI PT Organ of Corti ACCEPTED MANUSCRIPT OC SL 35,099 136,058 21 SG Array 28,833 100,024 58,955 232,182 14 374 46 7110 EP TE D M AN U SC RI PT 73,122 178,877 AC C Espn Kcne1 Kcnq1 LW ... performed laser- capture micro dissection in combination with next generation- sequencing analysis and determined the expression levels of all known EP deafness- associated genes in the organ of Corti,... in the other parts of the cochlea In the lateral wall, the Coch gene expression level was significantly higher than those in the other parts of the cochlea, while the gene expression levels of. .. PT Microscopic images of the laser- capture micro dissections of the mouse cochlea A, D: Microscopic view before laser cutting of the organ of Corti, B, E: Microscopic view after laser cutting of