biostudies-arrayexpressMarkus VogtHomo sapiensFastQC v0.12.1, cutadapt v1.18, STAR v2.7.10b, umi-tools v1.0.1, samtools v1.6GenomicAlignments v1.38.2, edgeR v4.0.16, R v4.3.3https://www.ebi.ac.uk/biostudies/studies/E-MTAB-163023' mRNA-seq was used to study the effects of USP7 degradation by the degraders NK266 and NK250 on gene expression in a human melanoma and PDAC cell line, respectively.biostudies-arrayexpressSequencing - Sequencing (100 cycles, single-end) was performed at the Lexogen sequencing service facility (Lexogen GmbH, Austria) which supplied demultiplexed FASTQ files for downstream analysis.Nucleic Acid Extraction - Following treatment, cells were lysed and total RNA was isolated using the AllPrep DNA/RNA Kit (Qiagen) in accordance with the manufacturer’s recommendations. RNA quality and integrity were evaluated using an Agilent TapeStation system.Sample Collection - The melanoma cell line Ma-Mel-47 was exposed to 1 µM NK266 and the PDAC cell line Panc89 to 1 µM NK250 against DMSO as a vehicle control in each case for 72 hours.Library Construction - Library preparation was carried out following the QuantSeq 3’ mRNA Library Prep Kit protocol (Lexogen). For each sample, 500 ng of RNA was used as input, and 15–20 PCR amplification cycles were performed, with the precise cycle number determined individually by qPCR. Library size distribution and concentrations were assessed on the Agilent TapeStation.OrganizationMINSEQE ScoreAssays and DataProcessed DataorganisationMAGE-TAB FilesSequence Alignment - Sequencing quality was checked using FastQC v0.12.1. QuantSeq Adapters and the polyA tail were trimmed using cutadapt v1.18. FASTQs were aligned to the GRCh38 reference genome using STAR v2.7.10b. Duplicate reads were removed based on unique molecular identifiers using umi-tools v1.0.1. BAM files were indexed using samtools v1.6.Data Transformation - Gene-wise reads were counted using the GenomicAlignments package v1.38.2 in R v4.3.3 and differential gene expression analysis was performed using edgeR v4.0.16.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsAgilent TapeStationElement AVITInoneProteolysis-targeting chimeras (PROTACs) co-op the ubiquitin system for targeted protein degradation, creating opportunities to interrogate cellular functions of proteins through "chemical knockdown". However, matched pairs of protein degraders and inhibitors, that possess high specificity and chemical complementarity, for individual components of the ubiquitin system have remained scarce. This includes reagents to modulate activity and abundance of deubiquitinases (DUBs). Here, using an integrated chemical biology approach, we explore cellular functions of the DUB USP7 as a case study by comparing inhibition and degradation in melanoma and pancreatic cancer cells. Through the synthesis of a degrader library, we identify and characterize potent USP7 PROTACs for each cancer type. Proteomic and cellular analyses reveal that selective USP7 degradation modulates both shared and distinct protein sets across both cancers without affecting cell growth. In contrast, prolonged inhibitor treatment induces USP7-independent proteomic and metabolic dysregulation, highlighting important caveats for the cellular use of hydroxypiperidine-based USP7 inhibitors. Collectively, our work provides a comprehensively characterized chemical toolbox to distinguish on-target phenotypes which will aid the understanding of USP7 in malignant diseases. More broadly, our data emphasize the importance of increased specificity via PROTAC-mediated degradation and the potential of this modality to elucidate cell-line specific functions of DUBs.RNA-seq of coding RNAHomo sapiensTargeted degradation of USP7 in solid cancer cells reveals distinct effects of deubiquitinase degraders and inhibitorsJohanna A. SeierBarbara M. GrünerMalte GerschNikolas KlinkSebastian UrbanNikolas Klink, Sebastian Urban, Johanna A. Seier, Bikash Adhikari, Martin P. Schwalm, Juliane Müller, Madeleine Dorsch, Farnusch Kaschani, Johannes Koch, Siska Führer, Markus Kaiser, Nina Schulze, Stefan Knapp, Elmar Wolf, Annette Paschen, Barbara M. Grüner, Malte GerschMarkus VogtAnnette Paschenfalse3' mRNA-seq of human Ma-Mel-47 (melanoma) and Panc89 (PDAC) cells treated with the USP7 degraders NK266 and NK250, respectively, for 72 h.3' mRNA-seq was used to study the effects of USP7 degradation by the degraders NK266 and NK250 on gene expression in a human melanoma and PDAC cell line, respectively.2026-03-27T00:00:00Z2026-05-18T13:25:52.826Z2025-11-27T00:09:37.583ZE-MTAB-16302ERP185702EFO_0002944EFO_0004170EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_000418410.1038/s41467-026-72295-x