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This dataset contains the numerical source data and uncropped Western blot images underlying the main and supplementary figures of the associated manuscript. The study investigates the use of PspCas13b guide RNAs (crRNAs) to recognize fusion-transcript breakpoint sequences, selectively disrupt oncogenic fusion RNAs, reduce fusion-protein expression, suppress downstream oncogenic signalling, and alter the survival and differentiation of fusion-dependent cancer cells. The workbook also contains short-read RNA-sequencing, Oxford Nanopore direct-RNA-sequencing, mass-spectrometry proteomics, flow cytometry, RT-qPCR, reporter-assay, and cell-proliferation data.

Description of the data and file structure

File included

• source_file_v3.xlsx: Excel workbook containing 75 worksheets. Each worksheet corresponds to a main-figure or supplementary-figure panel, as indicated by the worksheet name. Worksheets contain biological-replicate measurements, processed omics results, and/or uncropped source images.

Workbook-wide organization and conventions

• Each worksheet name identifies the corresponding figure panel (for example, Fig. 1a contains the source data for Figure 1a and suppl.Fig.6b contains the source data for Supplementary Figure 6b).
• Unless otherwise indicated, repeated numerical columns within the same experimental condition are independent biological replicates. Replicates may have blank column headers when the condition name is provided once across a group of adjacent columns.
• Values of 1 in normalized experimental datasets generally represent the value of the relevant control condition after normalization. Other values are fold values relative to that control and are unitless.
• Blank cells indicate that no value was available or applicable for that replicate or condition. Blank cells should not be interpreted as zero.
• Some worksheets contain embedded uncropped Western blot images. These images are retained within the corresponding worksheets to preserve their relationship to the sample names, treatment conditions, and quantitative data.
• Cell highlighting and other colour formatting are used only as visual aids to make sample names and experimental groups easier to identify. Highlighting does not encode statistical significance, inclusion/exclusion status, data quality, or any additional analytical variable.
• No analysis should rely exclusively on font colour, fill colour, highlighting, bold text, image placement, or other spreadsheet formatting.
• Rows containing individual gene or protein identifiers represent independent features, rather than biological replicates.
• Statistical tests and the number of biological replicates for each experiment are described in the associated manuscript and figure legends.

Common abbreviations, variables and interpretation keys

• ABL1, BCR, BCR::ABL1, EWSR1::FLI1, NPM::ALK, PAX3::FOXO1, RUNX1::RUNX1T1, SFPQ::ABL1, SNX2::ABL1 and SS18::SSX1/2: genes or oncogenic fusion transcripts/proteins studied.
• crRNA: CRISPR RNA. Numbered crRNAs are independent targeting guides.
• NT / crNT: non-targeting crRNA control.
• T / crRNA0: targeting crRNA condition. In experiments involving BCR::ABL1, crRNA0 targets the fusion breakpoint.
• OR: olfactory-receptor-targeting crRNA used as an additional negative control.
• WT: wild type. Depending on the worksheet, this denotes a fully matched guide or wild-type/parental BCR::ABL1 cells; the local worksheet heading and figure determine the meaning.
• T315I: BCR::ABL1 gatekeeper mutation conferring resistance to several tyrosine kinase inhibitors.
• SNP: single-nucleotide polymorphism/substitution introduced as a guide-target mismatch.
• MSM: mismatch; ranges such as 1-6nt MSM indicate the crRNA spacer nucleotide positions containing mismatches.
• -12 to +12: tiled crRNA positions relative to the fusion breakpoint; 0 denotes the guide centred on the breakpoint and negative/positive numbers indicate the positional offset in nucleotides.
• p190 / p210: BCR::ABL1 fusion-protein isoforms.
• Parental: parental K562 cells.
• TKI-resistant: K562 derivative resistant to tyrosine kinase inhibitor treatment and harbouring BCR::ABL1 T315I.
• DMSO: dimethyl sulfoxide vehicle control.
• Imatinib / Asciminib: ABL1-targeting small-molecule inhibitors.
• pSTAT5 / pERK: phosphorylated STAT5 or ERK; protein abundance or flow-cytometry signal was normalized to the relevant control.
• EGFP / GFP, mCherry, TagBFP / BFP and mFlame: fluorescent reporter proteins.
• MFI: mean fluorescence intensity, reported either in arbitrary fluorescence units or normalized to the relevant control.
• A.U.: arbitrary units.
• h: hours.
• N: number of independent biological experiments or biological replicates.
• SEM: standard error of the mean.
• CPM: counts per million sequencing reads.
• log2CPM: base-2 logarithm of counts per million.
• logFC / log2FoldChange / log2 FC: base-2 logarithm of the fold change. Positive values indicate higher abundance in the first-mentioned/test condition relative to the comparator; negative values indicate lower abundance.
• pvalue: unadjusted statistical test p-value.
• adjust-pvalue / adjusted P value: P value adjusted for multiple testing.
• FDR: false discovery rate. -log10(FDR) is the negative base-10 logarithm of the FDR; larger values represent stronger statistical evidence.
• gene: gene symbol or Ensembl gene identifier. Identifiers beginning ENSG are Ensembl identifiers, sometimes including a version suffix.
• protein: gene symbol or protein identifier representing a quantified protein group.
• Aberrant reads: reads containing a non-canonical junction or an internal gap/deletion defined by the analysis described in the manuscript.
• Contiguous/canonical reads: reads without the qualifying non-canonical junction or internal deletion.
• start / end: nucleotide coordinates of the beginning and end of an internal deletion or non-canonical junction interval in the relevant fusion-transcript reference sequence.
• In-frame split / out-of-frame split: aberrant junctions predicted to preserve or disrupt the coding frame, respectively.
• Fold Enrichment: observed enrichment of genes/proteins assigned to a pathway relative to the expected frequency.
• nGenes / number of Genes / gene count: number of submitted genes or proteins assigned to an enriched pathway.
• Pathway Genes: total number of genes annotated to the pathway in the reference set.
• Pathway: name of the enriched functional category.
• Genes: gene symbols contributing to the pathway enrichment.
• For binary pathway-membership matrices, 1 means that the gene/protein is assigned to the pathway and 0 means that it is not assigned.

Worksheet descriptions

Main Figure 1: silencing of fusion transcripts

• Fig. 1a: Normalized EGFP reporter fluorescence for tiled PspCas13b crRNAs targeting the BCR::ABL1-p190 breakpoint. Columns NT and -12 through +12 are guide conditions; rows 2-4 are biological replicates. Values are unitless and normalized to NT.
• Fig. 1b: Normalized EGFP reporter fluorescence for tiled crRNAs targeting SNX2::ABL1. Column and replicate interpretation is as for Fig. 1a.
• Fig. 1c: Normalized EGFP reporter fluorescence for tiled crRNAs targeting SFPQ::ABL1. Column and replicate interpretation is as for Fig. 1a.
• Fig. 1d: Normalized RT-qPCR abundance for BCR::ABL1-p190 across the tiled crRNA conditions. Values are normalized to NT; rows are biological replicates.
• Fig. 1e: Normalized RT-qPCR abundance for SNX2::ABL1 across the tiled crRNA conditions.
• Fig. 1f: Normalized RT-qPCR abundance for SFPQ::ABL1 across the tiled crRNA conditions.
• Fig. 1g: Normalized EGFP reporter fluorescence for tiled crRNAs targeting BCR::ABL1-p210.
• Fig. 1h: Source-image worksheet associated with Western blot assessment of BCR::ABL1-p190 silencing by tiled crRNAs. The corresponding quantitative context is provided by the adjacent Figure 1 worksheets and manuscript legend.
• Fig. 1j: Embedded uncropped Western blot images and associated source material for BCR::ABL1-p190, phosphorylated STAT5 and phosphorylated ERK in HEK293T cells under parental/control, NT, targeting-crRNA and imatinib conditions. Formatting/highlighting identifies samples only.

Main Figure 2: guide-target mismatch tolerance and fusion specificity

• Fig. 2a: Normalized reporter fluorescence for NT, fully matched (WT) and guides containing the indicated number of substitutions (3 SNP to 10 SNP) or a 14-nucleotide mismatch (14nt MSM). Rows are biological replicates; values are unitless and normalized to NT.
• Fig. 2b: Normalized reporter fluorescence for guides containing consecutive mismatches at the spacer positions specified in each header (for example, 1-6nt MSM). Rows are biological replicates.
• Fig. 2c: Embedded uncropped Western blot source images for the guide-target mismatch experiment, including BCR::ABL1-p190 and downstream signalling measurements.
• Fig. 2d: Normalized BCR::ABL1-mCherry reporter signal under NT, breakpoint-targeting crRNA0, crABL1 and crBCR conditions. Rows are biological replicates.
• Fig. 2e: Normalized ABL1-EGFP reporter signal under the same guide conditions as Fig. 2d.
• Fig. 2f: Normalized BCR-TagBFP reporter signal under the same guide conditions as Fig. 2d.
• Fig. 2 d,e,f WB: Uncropped Western blot source material corresponding to the protein-level specificity experiments in Figure 2d-f.

Main Figure 3: local cleavage and aberrant RNA products

• Fig.3a: Transcript-level short-read RNA-sequencing results obtained using a reference containing full-length BCR::ABL1. Column A contains gene/feature identifiers; log2CPM is average abundance; logFC is targeting versus NT fold change on the log2 scale. NT and T columns provide CPM values for the displayed BCR::ABL1 summary.
• Fig.3b: Short-read RNA-sequencing results obtained using a reference containing a 180-nucleotide region flanking the BCR::ABL1 breakpoint. Variables are as for Fig.3a; BCRABL1_breakpoint identifies the engineered breakpoint-region feature.
• Fig.3d: CPM and relative-frequency source data for aberrant versus contiguous BCR::ABL1 breakpoint reads in NT and targeting conditions. Adjacent repeated values are biological replicates. Relative frequencies are unitless proportions from 0 to 1.
• Fig.3e: Normalized RT-qPCR abundance measured with primer pairs at the breakpoint (Breakpoint primer), distal BCR::ABL1 positions (P2-P6), and EGFP. NT and crRNA0 each contain biological-replicate values; measurements are unitless and normalized to NT.
• Fig.3f: CPM values for the indicated NT and targeting groups used in the sequencing-based comparison; adjacent columns are biological replicates.
• Fig.3h: Start and end coordinates of internal deletions/non-canonical junctions detected by direct RNA sequencing. Coordinates are nucleotide positions in the BCR::ABL1 reference sequence. Group headings report the normalization denominator used for relative-frequency calculations.
• Fig.3i: Raw internal-deletion interval lengths/coordinate-derived values for NT and targeting direct-RNA-sequencing reads. The three adjacent columns under each condition contain values used for the displayed distributions.
• Fig.3j: Relative proportions of in-frame split reads, out-of-frame split reads, and canonical reads in NT and targeting conditions. Repeated columns are replicate or component values; values are unitless proportions from 0 to 1.
• Fig.3k: Relative proportions of aberrant and contiguous reads at the endogenous PAX3::FOXO1 breakpoint in Rh30 cells treated with NT, OR, crRNA1 or crRNA2. Four adjacent values are biological replicates; proportions range from 0 to 1.

Main Figure 4: oncogenic signalling and drug-resistant K562 cells

• Fig.4a: Normalized RT-qPCR abundance of BCR::ABL1-p210 fusion RNA, untranslocated BCR and untranslocated ABL1 in parental K562 cells treated with NT or breakpoint-targeting crRNA0. Adjacent values are biological replicates.
• Fig.4b: Normalized flow-cytometry MFI for pSTAT5 and pERK following imatinib treatment (DMSO versus Treated) or Cas13 targeting (NT versus crRNA0).
• Fig.4c: Embedded uncropped Western blot images and associated source material for BCR::ABL1-p210, untranslocated ABL1, pSTAT5 and pERK in parental K562 cells.
• Fig.4d: Alamar Blue proliferation measurements for parental K562 cells treated with DMSO or imatinib. Column A is time in hours; adjacent columns are biological replicates. Values are normalized, unitless proliferation/readout values.
• Fig.4e: Alamar Blue proliferation measurements for parental K562 cells treated with NT or BCR::ABL1-targeting crRNA0. Column A is time in hours; values are normalized and unitless.
• Fig.4g: Embedded uncropped Western blot images and quantification source material for wild-type and T315I BCR::ABL1-p190 silencing and signalling responses in HEK293T cells.
• Fig.4h: Embedded uncropped Western blot source images comparing imatinib responses in parental and TKI-resistant K562 cells.
• Fig.4i: Alamar Blue proliferation measurements for TKI-resistant K562 cells treated with DMSO or imatinib. Column A is time in hours; values are normalized and unitless.
• Fig.4k: Embedded uncropped Western blot images and quantification source material for PspCas13b delivery, BCR::ABL1-p210 silencing and tyrosine phosphorylation in K562 cells.
• Fig.4l: Embedded uncropped Western blot source images for BCR::ABL1-p210, pSTAT5 and pERK after targeting in parental and TKI-resistant K562 cells.
• Fig.4m: Alamar Blue proliferation measurements for parental and TKI-resistant K562 cells treated with NT or targeting (T) crRNA. Column A gives time in hours; adjacent columns are biological replicates; values are normalized and unitless.

Main Figure 5: transcriptomic and proteomic changes

• Fig.5b: Differential RNA-expression tables for parental and TKI-resistant K562 cells treated with Cas13 targeting crRNA versus NT. gene is the transcript identifier, the adjacent unlabeled column contains adjusted P values, and log2FoldChange is T relative to NT.
• Fig.5c: Differential RNA-expression tables for parental and TKI-resistant K562 cells treated with imatinib versus DMSO. gene, adjust-pvalue and log2FoldChange are defined above.
• Fig.5d: Matrix of log2 fold changes for 364 transcripts shared across the indicated Cas13 and imatinib comparisons. Column A contains gene identifiers; columns B-E contain log2 fold changes for the labelled comparisons.
• Fig.5e: Functional-enrichment scores for the pathway/category named in row 1 across the four comparisons named in row 2. Scores are transformed enrichment-significance values used to generate the figure; a value of 0 indicates no reported enrichment for that comparison.
• Fig.5f: Gene-by-pathway membership matrix for 32 common differentially expressed proteins. Column A is the gene/protein identifier; pathway columns use 1 for membership and 0 for non-membership; logFC is the mean protein log2 fold change across the relevant comparisons.
• Fig.5g: RNA and protein log2 fold changes for 16 genes detected confidently by both RNA sequencing and mass spectrometry. Column A contains gene identifiers. Columns B-E contain RNA log2 fold changes and columns F-I contain protein log2 fold changes for the comparisons stated in row 1.
• Fig.5h: Functional-enrichment results for the 16 shared RNA/protein genes. Columns are Enrichment FDR, number of Genes, Pathway Genes, Fold Enrichment, Pathway, Genes, and an additional transformed significance value used for plotting.

Main Figure 6: erythroid differentiation and rescue experiments

• Fig. 6b: Normalized CD235a MFI for K562 cells treated with Cas13 (NT versus T) or imatinib (DMSO versus Imatinib). Adjacent columns are biological replicates.
• Fig.6c: Embedded uncropped Western blot images and normalized quantification of BCR::ABL1-p190, BCR::ABL1-p210 and pSTAT5 in K562 cells overexpressing p190 and treated with NT or p210-targeting crRNA.
• Fig.6d: Time-course proliferation measurements for parental, MSCV-p190-overexpressing and MSCV-empty K562 cells treated with NT or targeting crRNA. Column A is time in hours; each labelled group contains three biological replicates. The final workbook header MSCV empty NT corresponds to the targeting group shown in the associated figure and should be interpreted with the sample labels embedded in the worksheet.
• Fig.6e: Normalized CD235a MFI for MSCV-empty and MSCV-p190 K562 cells treated with NT or targeting crRNA. Rows are biological replicates.

Main Figure 7: additional oncogenic fusion targets

• Fig. 7a: Normalized reporter fluorescence, embedded uncropped Western blot images and protein-quantification source data for RUNX1::RUNX1T1 targeted with crRNA1-crRNA5 versus NT.
• Fig. 7b: Normalized reporter fluorescence, embedded uncropped Western blot images and protein-quantification source data for NPM::ALK targeted with crRNA1-crRNA5 versus NT.
• Fig. 7c: Normalized reporter fluorescence, embedded uncropped Western blot images and protein-quantification source data for SS18::SSX1/2 targeted with crRNA1-crRNA5 versus NT.

Supplementary figures

• suppl. Fig. 2d: Normalized short-read RNA-sequencing abundance for NT and targeting conditions using the breakpoint-region reference. Column A contains gene/feature identifiers; NT and T contain log2-scale normalized abundance values used for the comparison/linear-regression analysis. Additional columns contain associated plotting values.
• suppl.Fig.2e: Differential-expression/volcano-plot source data for HEK293T cells expressing PspCas13b with targeting versus NT crRNA. Column A is the gene/feature identifier; logFC is the targeting-versus-NT log2 fold change; -log10(FDR) is the transformed statistical significance. No genes met the manuscript criteria of absolute log2FC > 1 and adjusted P value < 0.05.
• cas13d qpcr: Normalized RT-qPCR abundance of BCR::ABL1-p190-IRES-EGFP measured with a breakpoint primer, distal primer positions P2-P6, and an EGFP primer after RfxCas13d targeting. NT and T each contain three biological replicates.
• suppl.Fig.2f: CPM and relative-frequency source data for aberrant versus contiguous reads mapped across the full-length BCR::ABL1 transcript in HEK293T cells under NT and targeting conditions. Repeated columns are biological replicates; proportions range from 0 to 1.
• suppl.Fig.3h: Embedded uncropped Western blot images and normalized quantification of BCR::ABL1 and pSTAT5 after lipid-nanoparticle-mediated co-delivery of PspCas13b mRNA and either NT or targeting crRNA into K562 cells. Adjacent columns are three biological replicates.
• suppl.Fig.4b: Mass-spectrometry differential-abundance table used to assess proteomic changes after targeting. Column A contains protein/gene identifiers; log2FoldChange is targeting versus NT; -Log Student's T-test p-value TAR_NT is the negative logarithm of the unadjusted two-sided Student's t-test P value.
• suppl.Fig.4d: Quantification of EGFP mean fluorescence intensity in parental K562 cells at 48 and 72 h after nucleofection with PspCas13b and either NT or targeting crRNA. Values are fluorescence measurements; adjacent columns are three biological replicates.
• suppl.Fig.4e: Quantification of EGFP mean fluorescence intensity in TKI-resistant K562 cells at 48 and 72 h after nucleofection with PspCas13b and either NT or targeting crRNA. Values are fluorescence measurements; adjacent columns are three biological replicates.
• suppl. Fig 4f: Embedded uncropped Western blot images and normalized quantification of BCR::ABL1-p210 and pSTAT5 in parental and TKI-resistant K562 cells expressing PspCas13b with NT or targeting crRNA. These samples correspond to those assessed by flow cytometry in Supplementary Figure 4c.
• suppl.Fig.5c: CPM values for non-canonical split reads mapped exclusively to the BCR::ABL1 breakpoint in parental and TKI-resistant K562 cells treated with PspCas13b plus NT/targeting crRNA or with DMSO/imatinib. Adjacent values are biological replicates.
• suppl.Fig.6b: Gene Ontology enrichment results for the 400 transcripts shared among PspCas13b-treated parental and TKI-resistant K562 cells and imatinib-treated parental K562 cells. Columns are Enrichment FDR, nGenes, Pathway Genes, Fold Enrichment, Pathway, Genes, and an additional transformed significance value used for plotting.
• suppl.Fig. 6d: Gene Ontology enrichment results for selected differentially expressed transcripts. Variables are Enrichment FDR (adjusted P value), number of Genes (gene count), Pathway Genes, Fold Enrichment, Pathway, Genes, and an additional transformed significance value used for plotting.
• suppl.Fig.7a: Embedded uncropped Western blot images and normalized BCR::ABL1 protein quantification in parental and T315I/TKI-resistant K562 cells expressing PspCas13b with NT or targeting crRNA. These are the same lysates used for mass-spectrometry analysis; five biological replicates are provided.
• suppl. Fig. 7b: Differential protein-abundance results for parental and TKI-resistant K562 cells treated with Cas13 targeting crRNA versus NT. gene identifies the protein group, pvalue is the unadjusted P value, and log2FoldChange is T relative to NT.
• suppl. Fig. 7c: Differential protein-abundance results for parental and TKI-resistant K562 cells treated with imatinib versus DMSO. Variables are gene, pvalue and log2FoldChange.
• suppl. Fig. 7e: Matrix of protein log2 fold changes for 115 proteins differentially expressed at log2FC > 0.5 or < -0.5 in at least one PspCas13b-treated cell line. Column A contains protein/gene identifiers; columns B-E contain log2 fold changes for the Cas13 and imatinib comparisons named in row 1.
• suppl Fig. 8c: Gene Ontology enrichment categories and transformed enrichment-significance values for upregulated proteins in parental and TKI-resistant K562 cells treated with PspCas13b or imatinib. Column A is the functional category and column B is its plotting score; sequential table blocks correspond to the comparisons identified in the worksheet.
• suppl Fig. 8d: Gene Ontology enrichment categories and transformed enrichment-significance values for downregulated proteins in parental and TKI-resistant K562 cells treated with PspCas13b or imatinib. Variables are as for suppl Fig. 8c.
• suppl Fig. 9b: Matched RNA/protein log2 fold-change tables for parental and TKI-resistant K562 cells following Cas13 targeting or imatinib treatment. Each block contains a gene/protein identifier and paired protein and RNA logFC columns for the comparison named in row 1.
• suppl Fig.9c_x0009_: Matched Cas13-versus-imatinib log2 fold-change tables for parental and TKI-resistant transcriptomes and proteomes. Each block contains a feature identifier, logFC.cas13, and logFC.imatinib.
• suppl Fig.9d: Comparison of treatment responses between parental/WT and T315I cells. Separate blocks contain Cas13-induced RNA changes, imatinib-induced RNA changes, Cas13-induced protein changes and imatinib-induced protein changes. Each block contains a feature identifier plus logFC.WT and logFC.T315I.
• suppl Fig. 10a: Embedded uncropped Western blot images and normalized quantification of BCR::ABL1 and pSTAT5 in HEK293T cells expressing WT, C464W or T315I-C464W BCR::ABL1-p190 together with PspCas13b and NT or targeting crRNA. DMSO and 1 µM asciminib treatment controls are included. Adjacent columns are biological replicates.
• suppl Fig.10b: Normalized reporter data for EWSR1::FLI1 type 1, wild-type EWSR1 and wild-type FLI1 exposed to crRNAs with the indicated breakpoint-spanning designs (for example, crT1-22/8). The numbers separated by / indicate nucleotides complementary to the two fusion partners. Adjacent columns are biological replicates.
• suppl Fig.10c: Normalized reporter data for EWSR1::FLI1 type 2, wild-type EWSR1 and wild-type FLI1 exposed to the indicated type-2 breakpoint-spanning crRNAs. Variables are as for suppl Fig.10b.
• suppl.Figure 10d: Normalized RT-qPCR abundance of endogenous EWSR1::FLI1 type 2 in ES8 cells after delivery of PspCas13b mRNA with NT or targeting (T) crRNA. Rows are three biological replicates; values are unitless and normalized to NT.

Notes on data interpretation

• Normalized reporter, RT-qPCR, Western blot, flow-cytometry and proliferation values should be interpreted relative to the control stated in each worksheet and the corresponding figure legend.
• For omics tables, the comparison direction is written in the worksheet heading. For example, T VS NT means the fold change is calculated as targeting relative to non-targeting; imatinib vs DMSO means imatinib relative to vehicle.
• RNA-sequencing and proteomics tables may contain more features than are labelled in the published plots. The workbook provides the underlying processed values to enable reanalysis.
• The workbook includes processed source data rather than raw FASTQ or mass-spectrometry instrument files. Raw and processed omics data are available through the repositories listed below.

Sharing/Access information

Links to other publicly accessible locations of the data:

• Dryad dataset: https://doi.org/10.5061/dryad.0000000fh
• Short-read RNA-sequencing data, NCBI Gene Expression Omnibus accession GSE321679: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE321679
• Oxford Nanopore direct-RNA-sequencing data, NCBI BioProject accession PRJNA1417495: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1417495
• Mass-spectrometry proteomics data, ProteomeXchange/PRIDE accession PXD060758: https://www.ebi.ac.uk/pride/archive/projects/PXD060758
• Mass-spectrometry proteomics data, ProteomeXchange/PRIDE accession PXD074216: https://www.ebi.ac.uk/pride/archive/projects/PXD074216

Data were generated in this study and were not derived from an external dataset. Reference resources used for analysis included the human transcriptome/genome references GRCh37 version 19 and GRCh38 and the UniProt human reference proteome UP000005640.

Code/Software

No standalone analysis scripts or notebooks are included in this Dryad submission. Software used to generate or analyse the data included:

• ImageJ, including a laboratory-written batch macro, for fluorescence-image quantification.
• FlowJo version 10 for flow-cytometry analysis.
• GraphPad Prism version 9 for statistical analysis and graphing unless otherwise stated.
• Galaxy platform for short-read RNA-sequencing processing and alignment workflows.
• Arriba for fusion-transcript detection.
• IGV for alignment visualization.
• STAR version 2.7.10b for alignment used in breakpoint aberrant-read analysis.
• samtools for BAM indexing; samtools depth version 1.21 for direct-RNA-sequencing coverage.
• R version 4.2.2 for short-read aberrant-junction analysis.
• voom-limma version 3.42.2 for short-read RNA-sequencing differential-expression analysis.
• MinKNOW version 25.05.14 for Oxford Nanopore direct-RNA sequencing.
• dorado version 1.0.2 with model rna004_130bps_sup@v5.2.0 for Oxford Nanopore basecalling.
• minimap2 version 2.24 with the -ax splice option for direct-RNA read alignment.
• GenomicAlignments version 1.32.2 for extraction of splice junctions from direct-RNA alignments.
• R version 4.4.2, ggplot2 version 2.4.0, gggenes version 0.5.1 and cowplot version 1.2.0 for direct-RNA-sequencing visualization and figure assembly.
• MaxQuant version 2.2.0.0 with the Andromeda search engine for protein identification and label-free quantification.
• Perseus version 2.0.7.0 for proteomics filtering, log2 transformation and downstream analysis.
• STRING for protein-protein interaction network analysis.
• DAVID and ShinyGO for functional enrichment analyses.
• SRplot for chord and Sankey ontology plots.

Detailed experimental and computational procedures, statistical tests, and filtering thresholds are provided in the associated manuscript.