Vahts universal v8 rna seq library prep kit

The cochleae of wild-type and Gjb2^{35delG/35delG} mice were carefully collected and surgically divided into three equal sections, including the apical, middle, and basal turns. The total RNA of these sections was collected using a miRNeasy Micro Kit (QIAGEN, Cat#217084) following the manufacturer’s instructions. All collected RNA was used for library construction with the VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina (Vazyme, Cat# NR605). RNA-seq libraries were subjected to deep sequencing on an Illumina NovaSeq6000 platform following the manufacturer’s instructions (Illumina) to produce 150 bp paired-end reads by Genergy Biotechnology Co. Ltd. (Shanghai, China). Raw RNA-seq reads were cleaned using fastp (v0.20.0) and aligned to mm10 using STAR (v2.7.3a). Only unique mapped reads were kept and counted by featureCounts (v2.0.0), and FPKM (Fragments Per Kilobase of transcript per Million mapped fragments) and TPM (Transcripts Per Kilobase Million) were calculated by stringtie (v2.0). Genome coverage was counted by bamCoverage (v3.3.1). Differential gene expression was determined using DESeq2 (v1.30.0), and clusterProfiler (v3.18.1) was used for GO and KEGG analysis in R.

Total RNA was extracted from harvested EBs using TRIzol reagent (Invitrogen) following standard manufacturer’s instructions. 1 μg of total RNA was randomly fragmented and reverse-transcribed using the PrimeScript II 1st strand cDNA Synthesis Kit (TaKaRa, D6210A) according to the manufacturer’s instructions. VAHTS® Universal V8 RNA-seq Library Prep Kit for Illumina was used to prepare library for sequencing, about 60 million reads raw data were generated per sample from Novaseq 6000 PE150 equipment. Quality control, quality filtering, adapter trimming, and per-read quality pruning were performed using fastp^{55 (link)} (version: 0.23.2) and MultiQC^{69 (link)} (v 1.10.1). Filtered reads generated from RNA sequencing were aligned to the Mus musculus reference by STAR^{70 (link)} (v 2.7.4a). The “--quantMode” was set as GeneCounts to generate the count matrices. DESeq2^{71 (link)} (v1.30.1) were used to generate DEGs. Genes with an adjusted p value lower than 0.05 and absolute value of log₂(fold-change) greater than 0.5 were taken as DEGs. Seurat (version: 4.0.1) was used to evaluate the scores of gene sets. Of which, the CreateSeuratObject function was used to create objects, and we then used the SCTransform function to scale and normalize the expression matrix. The AddModuleScore was used to calculate the score of specific gene sets.

RNA libraries were constructed through ribosomal RNA depletion methods using Ribo-off rRNA Depletion Kit (Human/Mouse/Rat) (Vazyme #N406) and VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina (Vazyme #NR605). The libraries were sequenced on Illumina NovaSeq platforms with paired-end reads of 150 bp. The raw sequence data were demultiplexed and converted to FASTQ files with adapter and low-quality sequences quantified. Sequencing reads were aligned to the hg38 human reference genome. We obtained the Fragments Per Kilobase of transcript per Million mapped reads (FPKM) using StringTie (version 1.3.4) and Ballgown (version 2.14.149). To focus on genes with robust expression values for subsequent analysis, genes with FPKM of 0 in more than 30% samples were filtered out.