biostudies-arrayexpressKatherine WilliamsMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16526Single-cell RNA-seq data for Trex1 WT and KO mice from the tumor. Used for manuscript in submission at Science Advances.biostudies-arrayexpressSample Collection - Tumors were weighed and homogenized using a tumor dissociation kit (Miltenyi Biotec, 130-096-730) with a GentleMACS (Miltenyi Biotec, program 37°C _m_TDK1). Following procedures previously described (J. Lim, R. Rodriguez, K. Williams, J. Silva, A. G. Gutierrez, P. Tyler, F. Baharom, T. Sun, E. Lin, S. Martin, B. D. Kayser, R. J. Johnston, I. Mellman, L. Delamarre, N. R. West, S. Müller, Y. Qu, K. Heger, The Exonuclease TREX1 Constitutes an Innate Immune Checkpoint Limiting cGAS/STING-Mediated Antitumor Immunity. Cancer Immunol. Res. 12, OF1–OF10 (2024).), for scRNA-seq, cells were incubated with Fc receptor block and labeled with TotalSeq-C hashtag antibodies (clone M1/42;30-F11, BioLegend, hashtag 1-6) together CD11b (clone M1/70, BD Biosciences, RRID:AB_312798), CD19 (clone with the following surface markers: 6D5, BioLegend, RRID:AB_313654), CD45 (clone 30-F11, BD Biosciences, RRID:AB_2870247), CD90.2 (clone 30-H12, BioLegend, RRID:AB_313176), NK1.1 (Clone PK136, BioLegend, RRID:AB_389364). Subsequently, cells were labeled with calcein blue (Invitrogen, C1429) and 7-AAD (BioLegend, 420403). Samples were sorted into T cells (CD45+CD90.2+CD11b-NK1.1-CD19-) and non-T immune cells (CD45+CD90.2-CD11b+NK1.1+CD19+) with a FACSAria instrument (BD Biosciences). The same number of T cells or non-T immune cells from each mouse per each population were pooled and loaded into a Chromium single cell sorting system (10X Genomics).Library Construction - Reverse transcription (RT) was performed on the GEMs, and RT products were purified and amplified. DNA from hash tag oligos (HTO) was separated from cDNA based on size selection using SPRIselect beads (Beckman Coulter, B23318) and cDNA was used to generate Gene expression libraries. HTO libraries were generated using the 5' Feature Barcode Kit, (10X Genomics, PN-1000256). T cell receptor (TCR) libraries were amplified from the cDNA product (10X Genomics, PN 1000252) and enriched cDNA products were made into libraries using the library construction kit (10X Genomics, PN 1000190). Gene expression, TCR, and HTO libraries were then profiled using the Tapestation 4200 (Agilent Technologies) and quantified with Qubit Fluorometer (ThermoFisher Scientific).Sequencing - NovaSeqX+ (Illumina) was used to sequence the libraries. Gene Expression, HTO and V(D)J libraries were sequenced with 28 cycles for Read 1, 10 cycles for i7 index, 10 cycles for i5 index and 90 cycles for Read 2 with a targeted sequencing depth of 20,000, 2,000 and 5,000 read pairs per cell respectively.Nucleic Acid Extraction - Following procedures previously described (J. Lim, R. Rodriguez, K. Williams, J. Silva, A. G. Gutierrez, P. Tyler, F. Baharom, T. Sun, E. Lin, S. Martin, B. D. Kayser, R. J. Johnston, I. Mellman, L. Delamarre, N. R. West, S. Müller, Y. Qu, K. Heger, The Exonuclease TREX1 Constitutes an Innate Immune Checkpoint Limiting cGAS/STING-Mediated Antitumor Immunity. Cancer Immunol. Res. 12, OF1–OF10 (2024).), using the 10x Genomics Chromium Single Cell 5′ V2 Reagent Kit and Chromium Chip K Single Cell Kit, 4 samples of sorted single cell suspension were loaded onto the Chromium Controller. Cell counts and viability was checked using Cellometer K2 Image Cytometer cell count (Nexelom). Each sample was injected into microfluidic chips to form Gel Beads-in-Emulsion (GEMs) in the 10X Chromium X instrument.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - The resulting UMI counts were read into Seurat and TCR information was added to the metadata using scRepertoire(N. Borcherding, N. L. Bormann, G. Kraus, scRepertoire: An R-based toolkit for single-cell immune receptor analysis. F1000Research 9, 47 (2020).). Cells were filtered for singlets marked by DemuxEM. Cells were filtered for more than 500 genes expressed and less than 5% of reads from mitochondrial genes. After each of the described filtering steps, data was processed as follows: Data was log-normalized using the NormalizeData function from Seurat(Y. Hao, S. Hao, E. Andersen-Nissen, W. M. Mauck, S. Zheng, A. Butler, M. J. Lee, A. J. Wilk, C. Darby, M. Zager, P. Hoffman, M. Stoeckius, E. Papalexi, E. P. Mimitou, J. Jain, A. Srivastava, T. Stuart, L. M. Fleming, B. Yeung, A. J. Rogers, J. M. McElrath, C. A. Blish, R. Gottardo, P. Smibert, R. Satija, Integrated analysis of multimodal single-cell data. Cell 184, 3573-3587.e29 (2021).). The top 2,000 variable features were selected using FindVariableFeatures, and the data was scaled using ScaleData from Seurat. Principal components were determined using RunPCA, and nearest neighbors were calculated using FindNeighbors with 30 principal components (PCs). The data was clustered to a specified resolution using FindClusters with 30 PCs. UMAPs were generated with 30 PCs using RunUMAP. Cells were clustered with a resolution of 0.2, stromal cells were removed based on cluster marker gene expression and low score for immune cells coming from scGate scoring. scGate was rerun using the built-in generic model for CD4T and CD8T. Cells were reclustered at a resolution of 0.4 and clusters that had low fractions of CD8+ or CD4+ T cell calls from scGate along with low expression of Cd3d and cluster markers consistent with myeloid, gamma-delta T cells or NK cells were removed. Cells that were classified as CD8T from scGate were used as the final set of CD8+ T cells from the heart. T cell receptor encoding genes (Tra, Trb) were removed from both the tumor and heart objects to avoid biasing downstream analyses by clonotypes.Sequence Alignment - Raw sequencing data was demultiplexed by Illumina’s Bcl2Fastq software and resulting fastq files from each library were processed for analysis. Following procedures previously described (J. Lim, R. Rodriguez, K. Williams, J. Silva, A. G. Gutierrez, P. Tyler, F. Baharom, T. Sun, E. Lin, S. Martin, B. D. Kayser, R. J. Johnston, I. Mellman, L. Delamarre, N. R. West, S. Müller, Y. Qu, K. Heger, The Exonuclease TREX1 Constitutes an Innate Immune Checkpoint Limiting cGAS/STING-Mediated Antitumor Immunity. Cancer Immunol. Res. 12, OF1–OF10 (2024).), single-cell RNA-seq fastq files were processed through the count utility from CellRanger (G. X. Y. Zheng, J. M. Terry, P. Belgrader, P. Ryvkin, Z. W. Bent, R. Wilson, S. B. Ziraldo, T. D. Wheeler, G. P. McDermott, J. Zhu, M. T. Gregory, J. Shuga, L. Montesclaros, J. G. Underwood, D. A. Masquelier, S. Y. Nishimura, M. Schnall-Levin, P. W. Wyatt, C. M. Hindson, R. Bharadwaj, A. Wong, K. D. Ness, L. W. Beppu, H. J. Deeg, C. McFarland, K. R. Loeb, W. J. Valente, N. G. Ericson, E. A. Stevens, J. P. Radich, T. S. Mikkelsen, B. J. Hindson, J. H. Bielas, Massively parallel digital transcriptional profiling of single cells. Nat. Commun. 8, 14049 (2017).) (10X Genomics) using a custom reference generated from GENCODE M15 gene annotations and GRCm38/mm10. Multiplexed samples and antibody specific barcodes were parsed using a wrapper to the DemuxEM(48) package.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq XRNA-seq of coding RNA from single cellsMus musculusKatherine WilliamsfalsescRNA-seq from Trex1 KO in tumorSingle-cell RNA-seq data for Trex1 WT and KO mice from the tumor. Used for manuscript in submission at Science Advances.2026-03-25T00:00:00Z2026-03-25T02:03:18.563Z2026-01-14T16:10:25.225ZE-MTAB-16526ERP187583EFO_0002944EFO_0004170EFO_0005684EFO_0004917EFO_0005518EFO_0003816EFO_0004184