biostudies-arrayexpressMaria PuschhofHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16843Human colorectal cancer organoids were treated with the TROP2-targeting antibody-drug conjugate Sacituzumab Govitecan (SG) or non-targeting control IgG1-SN-38. Samples were harvested at different time points under treatment {0h, 3h, 6h, 9h, 12h} as well as after drug washout at 12 h on the following time points {12h + 12h, 12h + 36h, 12h + 60h}. Samples were subjected to single-cell RNA-seq using sample hashtag multiplexing.biostudies-arrayexpressNucleic Acid Extraction - Hashtag oligo-labelled samples were pooled (up to 12,000 cells each) and processed for single-cell RNA-sequencing using the 10X Chromium Next GEM Single Cell Reagent Kit 3.1 according to the manufacturer’s instructions (Dual Index protocol). First, cells are resuspended in a master mix and loaded onto a chip. After encapsulation, cells were lysed inside their GEMs.Sequencing - Next generation sequencing was performed at the German Cancer Research Center using an Illumina NovaSeq X Plus 10B instrument (paired-end, two lanes per library).Sample Collection - Organoids were harvested and washed twice with cold PBS (1500 rpm, 4 °C, 5 min). Dissociation of the organoids was achieved resuspending the pellet in TrypLE (Thermo Fisher Scientific, catalog #12605036) and incubated at 37 °C in a water bath until a homogeneous cell suspension was obtained, pipetting the mix occasionally to mechanically accelerate the dissociation process. To stop the reaction, PBS + 2% FCS was added to the solution and spin down (1 500 rpm, 4 °C, 5 min). Eventually, cells were cryopreserved until all timepoints were collected for further processing. For sample multiplexing, frozen samples were rapidly thawed in a water bath 37 °C and transferred to a conical tube with added fresh organoid medium + 2% FBS and spin down. Cell pellet was then resuspended in 100 µL of PBS + 2% FCS with 1:50 FcR Blocking Reagent (Miltenyi Biotec, catalog #130-059-901) and stained with 1µL of hashing antibodies (Total-Seq-B, BioLegend, 0.5 µg antibody) for 30 minutes at 4 °C. Afterwards, cells were washed twice with PBS + 2% FCS. Cell pellet was then resuspended in PBS + 2% FCS added with a live/dead marker (DAPI, Invitrogen catalog # 62248 or Zombie NIR, BioLegend, catalog # 423106), filtered, and multiplexed samples were sorted for live cells in a single tube.Library Construction - After cell lysis inside of GEMs, mRNA is reverse transcribed to cDNA with droplet-specific barcodes. After GEM breakdown, the library is being prepared according to the protocol including cDNA amplification by PCR in 11 cycles.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Quality control and filtering was performed at sample level. First, the fraction of mitochondrial reads (scanpy.pp.calculate_qc_metrics) and the doublet probability per cell barcode (scanpy.external.pp.scrublet) was computed. Cells were filtered for having at least 1,200 genes, while genes were filtered for being expressed in at least 3 cells. Finally, cells with a gene count exceeding the 99.5 % percentile or with a mitochondrial fraction larger than 20 % or with a doublet score larger than 0.25 were excluded. Count matrices of high quality cells were concatenated (across samples), normalized and log1p transformed for downstream analysis.Sequence Alignment - Reads were mapped to the human reference genome assembly refdata-gex-GRCh38-2020-A and demultiplexed into their original samples using the cellranger multi pipeline with default settings of the 10X Genomics Cell Ranger (v9.0.1).UnknownTranscriptomicsIllumina NovaSeq XRNA-seq of coding RNA from single cellsHomo sapiensTROP2 targeting reveals therapy-driven cell state dynamics and vulnerabilities in colorectal cancerNuria Vaquero-Siguero, Nikolaos Georgakopoulos, Maria C Puschhof, Ioannis Chiotakakos, Jasmin Meier, Sigrid K Fey, Gabriele Diamante, Manuel Mastel, Aitana Guiseris Martinez, Guillaume Belthier, Nikolai Schleußner, Julia Volk, Carolin Artmann, Bryce Lim, Ronald Koschny, Cyrill Wehling, Kyanna S Ouyang, Michael Günther, Solveig Kuss, Paula Hoffmeister, Moritz Mall, Jens Neumann, Steffen Ormanns, Martin Schneider, Thomas Schmidt, Jens Puschhof, Andreas Trumpp, Jacco van Rheenen, Julio Saez-Rodriguez, Bruno C Köhler, Rene JackstadtMaria PuschhoffalseTime course analysis of human colorectal cancer organoid treatment with Sacituzumab Govitecan (SG) or untargeted control IgG1-SN-38Human colorectal cancer organoids were treated with the TROP2-targeting antibody-drug conjugate Sacituzumab Govitecan (SG) or non-targeting control IgG1-SN-38. Samples were harvested at different time points under treatment {0h, 3h, 6h, 9h, 12h} as well as after drug washout at 12 h on the following time points {12h + 12h, 12h + 36h, 12h + 60h}. Samples were subjected to single-cell RNA-seq using sample hashtag multiplexing.2026-04-01T00:00:00Z2026-04-01T11:27:06.62Z2026-04-01T11:25:40.897ZE-MTAB-16843ERP191666EFO_0002944EFO_0004170EFO_0005684EFO_0004917EFO_0005518EFO_0003816EFO_0004184