Novaseq 6000 instrument

Isolated RNA was sent for next-generation sequencing to GenomeScan B.V. (Leiden, The Netherlands). The RNA integrity was determined using a Fragment Analyzer system (Agilent) or a 2100 Bioanalyzer (Agilent). Samples were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (catalog number E7760S/L; New England BioLabs [NEB]) according to the instructions of the manufacturer. Briefly, host poly(A) mRNA was isolated from total RNA using oligo(dT) magnetic beads. After the fragmentation of the mRNA, cDNA was synthesized, sequencing adapters were ligated, and the fragments were PCR amplified. The quality and yield of the sequencing library were determined using a Fragment Analyzer (Agilent). The resulting products had a size distribution with a broad peak between 300 and 500 bp. Clustering and sequencing of 1.1 nM DNA samples were performed using an Illumina NovaSeq 6000 instrument with NovaSeq control software NCS version 1.7, according to the instructions of the manufacturer. The sequence length of the short reads ranged between 151 and 159 bp.

Total RNA sequencing was performed at the NGS Competence Centre at IKMB (Kiel, Germany) from the same aliquots of total RNA that were also used for small RNA sequencing. Libraries were prepared using the TruSeq stranded total RNA kit (Illumina), according to the manufacturer’s instructions, and sequenced on a NovaSeq 6000 instrument (Illumina) with 2 × 100 bp reads. Reads were pseudoaligned to the human transcriptome (Ensembl v. 100) [23 (link)] using kallisto v0.46.1 [24 (link)]. To account for intra-sample technical variance, 100 bootstraps were performed per biological replicate. Raw reads were normalized to transcripts per million (TPM) considering protein-coding isoforms of protein-coding genes and lncRNA isoforms of lncRNA genes only. Isoforms with less than 6 assigned raw reads and/or less than 0.1 TPM in more than 80 % of all replicates in either condition were excluded from the analysis. Additional filtering, normalization and differential gene expression analysis was carried out using the R package sleuth (version 0.30.0) [25 (link)] adjusting for age at death, sex, RNA integrity (RIN), post-mortem-interval (PMI), and the first 10 principle components of the underlying expression profiles to account for additional undetected confounding.

After euthanasia, total RNAs from mouse hearts at the ages of 1, 12, and 36 weeks were extracted using the RNeasy Plus Universal Mini Kit (Qiagen). Nine representative libraries were generated for the nine groups (wild-type, heterozygote, and homozygote of 1 week, 12 weeks, and 36 weeks) by RNA-seq with SMART-seq v4 Ultra Low Input RNA kit for sequencing (Takara Bio Inc.) and Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA) and sequenced on a NovaSeq 6000 instrument (Illumina).

Total RNA was extracted from BRAFi‐sensitive or resistant SK28, Mel501, and M229, cells before and after knockout out of AhR using the NucleoSpin RNA kit (Macherey Nagel, Düren, Germany). A complementary DNA library was prepared and sequencing performed according to the Illumina standard protocol by Beijing Novel Bioinformatics Co., Ltd. (https://en.novogene.com/). RNAseq was performed in collaboration with Novogene (Beijing, China). Libraries were generated from 500 ng total RNA using a Truseq Stranded mRNA kit (Illumina). The concentration of the library was first determined using a Qubit2.0 fluorimter and then diluted to 1 ng/μl. The size of the insert was checked using an Agilent bioanalyzer and further quantified by qPCR (library concentration > 2 nM). An aliquot (0.5 nM) of the pool was loaded on a high‐output flow cell and sequenced on a NovaSeq 6000 instrument (Illumina) with 2 × 150 bp paired‐end chemistry in two runs. Reads were aligned to human genome release hg38 using HISAT2 V2.0.5 with default parameters. Quantification of the expressed genes was performed using CUFFDIFF v2.2.1. The quality of the RNA‐Seq count data was assessed using the Novogene standard protocol. The RNA‐Seq data presented in this article was submitted to the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under the accession number (GSE166617).