The samples. The cDNAs were synthesized from the total RNAs for

The samples. The cDNAs were synthesized from the total RNAs for each sample with the Clontech SMARTerTM Ultra Low RNA kit. A sequencing library was prepared from each sample using an Illumina Paired End Sample Prep kit according to the manufacturer’s protocol. The average insert size of the libraries was 124 bp. Each cDNA library was sequenced in a 50cycle single read flow cell lane on an Illumina HiSeq 2000 in the Next-Generation Sequencing and Expression Analysis Core at the University at Buffalo. Four Relebactam chemical information biological repeats were performed for the mouse samples and three biological repeats were performed for the rat samples. For the RNA-sequencing data analysis, the mouse sequence reads were aligned to the mouse reference genome sequence and the rat sequence reads were aligned to the rat genome sequence using TopHat version 1.3.2 and Bowtie. The resulting alignments were further assembled and annotated using Cufflinks software. The abundance of gene expression was normalized to the fragments per kilobase of transcript per million mapped reads . Finally, the program, Cuffdiff, was used to define the genes and transcripts that were differentially expressed after MedChemExpress Indirubin-3′-oxime acoustic overstimulation. 2.6. Functional annotation of the differentially expressed genes The Database for Annotation, Visualization, and Integrated Discovery v.6.7 and Ingenuity Pathway Analysis were used to determine the functional relevance of the differentially expressed genes. DAVID is a web-based tool for identifying the enriched gene ontology terms and molecular functions associated with the genes of interest. The Kyoto Encyclopedia of Genes and Genomes database accessed via DAVID was used to analyze the molecular pathways. To reduce the false positive identification, attention was placed on the captures that had a P value or a false discovery rate < 0.05. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Hear Res. Author manuscript; available in PMC 2017 March 01. Yang et al. Page 6 IPA was used to define the potential upstream transcriptional regulators for the differentially expressed genes. This analysis predicts the genes, molecules, and drugs that could regulate the genes of interest based on the available knowledge of the role of these molecules in downstream regulation. The predictions of the activation and the inhibition states of transcriptional regulators were made based on the direction of the changes in the expression of the downstream genes. The activation Z-score was used to identify the most relevant regulators. This score infers the likely activation states of the upstream regulators based on comparison with a model that assigns random regulation directions. An absolute Z-score of greater than or equal to 2 was used to identify the upstream regulators. 2.7. Protection of one ear from acoustic overstimulation for the mouse RNA-sequencing experiment For the mouse RNA-sequencing experiment, we used intra-subject comparison to reduce the influence of systemic variation. Specifically, one ear of the subject was exposed to noise, and the other ear was protected from the noise and served as the control. The protection was achieved by disrupting the conductive transmission function of the outer and the middle ear. Specifically, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1985460 the animal was anesthetized, and silicon grease was gently pushed into the ear canal to fill the canal and middle ear cavity using a syringe with a fine needle under a surgical microscope. This procedure resulted in a si.The samples. The cDNAs were synthesized from the total RNAs for each sample with the Clontech SMARTerTM Ultra Low RNA kit. A sequencing library was prepared from each sample using an Illumina Paired End Sample Prep kit according to the manufacturer’s protocol. The average insert size of the libraries was 124 bp. Each cDNA library was sequenced in a 50cycle single read flow cell lane on an Illumina HiSeq 2000 in the Next-Generation Sequencing and Expression Analysis Core at the University at Buffalo. Four biological repeats were performed for the mouse samples and three biological repeats were performed for the rat samples. For the RNA-sequencing data analysis, the mouse sequence reads were aligned to the mouse reference genome sequence and the rat sequence reads were aligned to the rat genome sequence using TopHat version 1.3.2 and Bowtie. The resulting alignments were further assembled and annotated using Cufflinks software. The abundance of gene expression was normalized to the fragments per kilobase of transcript per million mapped reads . Finally, the program, Cuffdiff, was used to define the genes and transcripts that were differentially expressed after acoustic overstimulation. 2.6. Functional annotation of the differentially expressed genes The Database for Annotation, Visualization, and Integrated Discovery v.6.7 and Ingenuity Pathway Analysis were used to determine the functional relevance of the differentially expressed genes. DAVID is a web-based tool for identifying the enriched gene ontology terms and molecular functions associated with the genes of interest. The Kyoto Encyclopedia of Genes and Genomes database accessed via DAVID was used to analyze the molecular pathways. To reduce the false positive identification, attention was placed on the captures that had a P value or a false discovery rate < 0.05. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Hear Res. Author manuscript; available in PMC 2017 March 01. Yang et al. Page 6 IPA was used to define the potential upstream transcriptional regulators for the differentially expressed genes. This analysis predicts the genes, molecules, and drugs that could regulate the genes of interest based on the available knowledge of the role of these molecules in downstream regulation. The predictions of the activation and the inhibition states of transcriptional regulators were made based on the direction of the changes in the expression of the downstream genes. The activation Z-score was used to identify the most relevant regulators. This score infers the likely activation states of the upstream regulators based on comparison with a model that assigns random regulation directions. An absolute Z-score of greater than or equal to 2 was used to identify the upstream regulators. 2.7. Protection of one ear from acoustic overstimulation for the mouse RNA-sequencing experiment For the mouse RNA-sequencing experiment, we used intra-subject comparison to reduce the influence of systemic variation. Specifically, one ear of the subject was exposed to noise, and the other ear was protected from the noise and served as the control. The protection was achieved by disrupting the conductive transmission function of the outer and the middle ear. Specifically, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1985460 the animal was anesthetized, and silicon grease was gently pushed into the ear canal to fill the canal and middle ear cavity using a syringe with a fine needle under a surgical microscope. This procedure resulted in a si.