Nextseq 500

The RNA-seq method used in the present analysis has been previously published19 (link) but briefly, the Illumina NextSeq 75 bp High Output sequencing kit with Illumina NextSeq 500 (Illumina, San Diego, California; 75 cycle, single read sequencing) were used according to the manufacturer-recommended procedure. Samples were run on the Illumina NextSeq 500 for single-end sequencing and transferred to the Illumina Base Space interface. Quality control (QC) was performed by assessing the numbers of reads in millions passing filter and the per cent of indexed reads. All runs passed the Illumina QC procedures. The clean reads were mapped with the star aligner to the GENCODE V.27 of the transcriptome and hg38 human genome build. The HTseq20 (link) software was used to count aligned reads mapping to each gene.

DNA from Gl261 tumor tissue from R and NR mice was extracted using the INVISORB^® DNA Tissue Mini Kit (STRATEC Biomedical AG) according to the manufacturer’s instruction. RNA contamination was eliminated by RNase digestion with 10 mg ml⁻¹ RNase at room temperature (RT) for 5 min (Sigma-Aldrich). Exome sequencing was performed on the Illumina NextSeq500 platform (Illumina Inc, San Diego, Calif.) using High output flow cell (75 nt reads paired end + 8 nt index). SureSelectXT Target Enrichment System (Agilent Technologies) was used for library generation according to the manufacturer’s instructions. To convert the vendor-specific sequencing data format generated by the Illumina NextSeq500 to a standard file format, the Illumina tool bcl2fastq (v2.15.0.4)⁴⁶ was used. To check the sequencing read quality, reports were generated with the tool fastqc (v0.10.1)⁴⁷ . After quality checks the alignment was performed with bwa mem (v0.7.5)⁴⁸ and the mouse reference genome GRCm38.68. The picard-tools (v1.105)⁴⁹ were used to remove duplicates from the alignment files. The sorting and indexing of these files was done with samtools (v0.1.19)^{50 (link)}. Afterward the variants were called by samtools mpileup (v0.1.19) for single-nucleotide variants and platypus (v0.7.9.1)⁵¹ for insertions and deletions. The basic annotations of the called variants was done with annovar (v2013-08-23)^{52 (link)}.

NGS analysis was performed as follows: RNA was isolated from FFPE tissue sections using the Qiagen miRNeasy FFPE kit (Qiagen, Hilden, Germany) and purified using RNeasy MinElute cleaning (Qiagen). The total RNA concentration was measured using the Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), and the quality was checked using the 2100 Bioanalyzer with the RNA 6000 Nano kit (Agilent Technologies, Santa Clara, CA, USA). Library preparation was performed using QIAseq stranded total RNA library kit (Qiagen) with QIAseq Fast Select RNA Removal kit (Qiagen) for RNA depletion according to the manufacturer’s instructions and quality assessed by 2100 Bioanalyzer (Agilent). The libraries were run on the Illumina NextSeq 500 (Illumina) platform using NextSeq 500/550 High Output Kit v2.5 (75 Cycles) according to the manufacturer’s instructions. The analysis of the RNA sequencing data was processed using the Archer Analysis bioinformatics platform (v 6.2).

DNA and RNA from fresh frozen biopsies, blood and tumor-infiltrating lymphocytes were extracted as described. Primary eye tumors were sectioned and processed using an FFPE DNA kit (Qiagen). Whole-genome DNA libraries were made using the Illumina TruSeq PCR-free kit or Illumina TruSeq Nano in some cases where low input material was available and sequenced with Illumina HiSeq X Ten at SciLifeLab in Stockholm or Illumina NovaSeq 6000 at GeneCore SU in Gothenburg. RNA libraries were made using the Illumina TruSeq Stranded mRNA kit with poly-A selection and sequenced with Illumina HiSeq 2500 at SciLifeLab in Stockholm or with Illumina NextSeq 500 at GeneCore SU in Gothenburg. Exome-sequencing libraries were prepared with the NextSeq 500 HighOutput Kit v2 and sequenced with Illumina NextSeq 500.

For cellular RNA-seq with USP30-AS1 knockdown, total RNA was isolated and DNase I was treated using the Maxwell Simply RNA Kit (Promega). 1 μg of quality-verified RNA was used for library preparation and sequenced on Illumina Nextseq 500 (75 bp single-ended mode, 1 × 10⁶ reads/sample). 1 μg of quality-verified RNA was used for library preparation and sequenced on Illumina Nextseq 500 (75 bp single-ended mode, 1 × 10⁶ reads/sample). Differential expression analysis performed using edgeR56 in R/Bioconductor, and the following filters were used to select important genes: adjusted p value < 0.05 and |log₂FC|> 0.585.

scATAC-seq libraries were prepared using the Chromium Next GEM single-cell ATAC reagent kit v1.1 following the manufacturer’s instructions with the modifications described in the ASAP-seq and mtscATAC-seq protocols to retrieve TotalSeq antibody-derived tags (ADTs), hashtag oligonucleotides (HTOs) and mtDNA^{16 (link)–18 (link)}. The saved eluate from the silane bead elution and the supernatant of the first SPRIselect purification were combined for amplification of ADT/HTO libraries using the KAPA HiFi ready mix (Roche) with sample-specific index primers (Illumina small RNA RPIx/Truseq D7xx) and purified using SPRIselect reagent. Library size and quality were analyzed using a Fragment Analyzer (Advanced Analytical) before fragmentation and after final purification. Final library concentrations were measured on a Qubit 2.0 fluorometer (Thermo Fisher). Libraries were sequenced on a NextSeq500 or NovaSeq6000 sequencer (Illumina) using longer read1/2 configurations than suggested by 10x Genomics to improve mitochondrial genotyping (NextSeq500: R1 72 cycles, R2 72 cycles, I1 8 cycles, I2 16 cycles; NovaSeq6000: R1 88 cycles, R2 88 cycles, I1 8 cycles, I2 16 cycles).