Truseq stranded mrna library kit for neoprep

Liver transcriptomics were performed using RNA sequencing on RNA extracts from terminal liver samples (15 mg fresh tissue), as described in detail elsewhere.²¹ RNA sequence libraries were prepared with NeoPrep (Illumina, San Diego, CA) using Illumina TruSeq stranded mRNA Library kit for NeoPrep (Illumina) and sequenced on the NextSeq 500 (Illumina) with NSQ 500 hi‐Output KT version 2 (75 CYS; Illumina). Reads were aligned to the GRCm38 version 84 Ensembl Mus musculus genome using STAR version 2.5.2a with default parameters. A lower detection limit for gene expression was defined based on raw mapped read counts (RPKM of 0.1). The R‐package DESeq2 was used for differential gene expression analysis. Gene set analysis was conducted with the R package PIANO version 1.18.1 using the Stouffer method, and p values were corrected for multiple testing using the Benjamini‐Hochberg method (5% false discovery rate < 0.05). RNA sequencing data from the pharmacological intervention studies are accessible using a web‐based global gene expression data viewer (Gubra Gene Expression Experience [GGEX], https://rnaseq.gubra.dk/).

Liver transcriptome analysis was performed by RNA sequencing on RNA extracts from terminal liver samples (15 mg fresh tissue), as described in detail elsewhere[22 (link),23 (link)]. The RNA quantity was measured using Qubit^® (Thermo Scientific, Eugene, OR, United States). The RNA quality was determined using a bioanalyzer with RNA 6000 Nano kit (Agilent, Waldbronn, Germany). RNA sequence libraries were prepared with NeoPrep (Illumina, San Diego, CA, United States) using Illumina TruSeq stranded mRNA Library kit for NeoPrep (Illumina, San Diego, CA, United States) and sequenced on the NextSeq 500 (Illumina, San Diego, CA, United States) with NSQ 500 hi-Output KT v2 (75 CYS, Illumina, San Diego, CA, United States). Reads were aligned to the GRCm38 v84 Ensembl Mus musculus genome using STAR v.2.5.2a with default parameters[38 (link)]. Differential gene expression analysis was performed with DEseq237. Genes with a Benjamini and Hochberg adjusted P ≤ 0.05 (5% false discovery rate, FDR) were regarded as statistically significantly regulated. The Reactome pathway database[39 (link)] was used as gene annotation in a gene set analysis using the R package PIANO v.1.18.1[40 (link)], with the Stouffer method and Benjamini-Hochberg adjusted P values (FDR < 0.01).

Hepatic transcriptome analysis was performed by RNA sequencing on RNA extracts from terminal liver samples (15 mg fresh tissue), as described in detail previously[19 (link)]. The RNA quantity was measured using Qubit^® (Thermo Scientific, Eugene, OR, United States). The RNA quality was determined using a bioanalyzer with RNA 6000 Nano kit (Agilent, Waldbronn, Germany). RNA sequence libraries were prepared with NeoPrep (Illumina, San Diego, CA, United States) using Illumina TruSeq stranded mRNA Library kit for NeoPrep (Illumina, San Diego, CA, United States) and sequenced on the NextSeq 500 (Illumina, San Diego, CA, United States) with NSQ 500 hi-Output KT v2 (75 CYS, Illumina, San Diego, CA, United States). Reads were aligned to the GRCm38 v84 Ensembl Mus musculus genome using STAR v.2.5.2a with default parameters[36 (link)]. Differential gene expression analysis was performed with DEseq2[37 (link)]. Genes with a Benjamini and Hochberg adjusted P ≤ 0.05 (5% False Discovery Rate) were regarded as statistically significantly regulated. Enrichment analysis of KEGG pathways were performed using the clusterProfiler package for R[38 (link)].