Computational protocol: Direct Reprogramming of Spiral Ganglion Non-neuronal Cells into Neurons: Toward Ameliorating Sensorineural Hearing Loss by Gene Therapy

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Protocol publication

[…] RNA was extracted using the Single Cell RNA Purification Kit (NORGEN, #51800) from each of the following groups; Tau-EGFP positive endogenous PANs, DsRed (Ascl1 and NeuroD1) and Tau-EGFP positive iN, and DsRed positive vector-control (VC). The quality of extracted RNA was verified by Bioanalyzer 2100 RNA 6000 pico chip (Agilent Technologies) and the concentration was measured by Qubit RNA HS Assay (Thermo Fisher). RNA library preparation was performed using a two-pronged approach: (1) Two ng of input RNA was converted to double stranded cDNA using Clontech SMARTer Ultra Low Input RNA Kit v3 using Clontech's proprietary Switching Mechanism at 5' End of RNA Template (SMART) technology, following the manufacturer's instructions; double stranded (ds) DNA was then quantified by Qubit HS assay and then (2) 1 ng of ds-DNA was used as input material for the Nextera XT library preparation following Illumina's recommended protocol. One microliter of the final RNA-Seq libraries was loaded on a Bioanalyzer 2100 DNA High Sensitivity chip to check for size; RNA libraries were quantified by qPCR using the Kapa Library Quantification Illumina/ABI Prism Kit protocol (KAPA Biosystems). Libraries were pooled in equimolar quantities and paired-end sequenced on an Illumina HiSeq 2500 platform using a High Throughput Run Mode flowcell and the V4 sequencing chemistry following Illumina's recommended protocol to generate paired-end reads of 126-bases in length. Three biological repeats of each group were loaded and sequenced at 40M reads. We mapped FASTQ files to mm10 genome by HISAT2 protocol (Kim et al., ; Pertea et al., ), which is a refined protocol based on TopHat2 (Kim et al., ). Expression data were normalized by the transcripts per million reads (TPM) method (Wagner et al., ), which enables us to avoid statistical biases inherent in the traditional read per kilobase per million reads (RPKM) method (Mortazavi et al., ). Genes were included in our analysis if at least one of the three samples surpassed a threshold of 0.2 TPM. Using this method, we detected a total of 17,601 genes. Raw RNA-seq data have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession number GSE107461. […]

Pipeline specifications

Software tools HISAT2, TopHat
Databases GEO
Application RNA-seq analysis
Chemicals Puromycin Aminonucleoside