Computational protocol: Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity

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Protocol publication

[…] Rat livers were collected 24 h post-50 mg/kg single dose of GalNAc-siRNAs and snap-frozen. Rat hepatocytes (BioreclamationIVT) were transfected with 10 nM GalNAc-siRNAs using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to manufacturer’s instructions, and harvested 24 h post-transfection. Rat hepatocytes were not tested for mycoplasma contamination. RNA extracted with the miRNeasy kit (Qiagen) was used for cDNA library preparation with the TruSeq Stranded Total RNA Library Prep Kit (Illumina) and sequenced on the HiSeq or NextSeq500 sequencers (Illumina), all according to manufacturers’ instructions. Raw RNAseq reads were filtered with minimal mean quality scores of 25 and minimal remaining length of 36, using fastq-mcf. Filtered reads were aligned to the Rattus norvegicus genome (Rnor_6.0) using STAR (ultrafast universal RNAseq aligner) with default parameters. Uniquely aligned reads were counted by featureCounts. Differential gene expression analysis was performed by the R package DESeq2.The open source DESeq2 R package was used for the RNAseq data analysis. […]

Pipeline specifications

Software tools ea-utils, Subread, DESeq2
Application RNA-seq analysis
Organisms Rattus norvegicus
Diseases Drug-Related Side Effects and Adverse Reactions