biostudies-arrayexpressJeffrey IshizukaHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15917We developed PERCEPT, an approach that uses ex vivo perturbational single-cell RNA sequencing to compare the response of immunomodulatory treatments with unstimulated controls directly in patient samples. Using PERCEPT, we tested cytokines and innate immune agonists in melanoma and Merkel cell carcinoma (MCC) and identified the dsRNA mimetic, RIG-I agonist, Stem Loop RNA (SLR) 14 as a powerful inducer of anti-viral states and enhancer of T cell activation. Our results demonstrate the utility of high-dimensional controlled perturbation of patient samples to identify mechanisms of innate immune response and resistance.biostudies-arrayexpressNucleic Acid Extraction - Sorted cells were immediately loaded into a 10x Chromium reaction.Library Construction - Chromium Next GEM Single-Cell 5′ kits v.2 were used to generate GEX, VDJ and Cite-seq libraries according to the manufacturer’s protocol.Sequencing - Sorted cells were then resuspended to 700-1200 cells per ul and barcoded for multiplexed single cell sequencing using 10x Genomics 5’v2 chemistry (10x Genomics, PN-1000263).Sample Collection - Humor tumor samples were collected from patients. Resected human tumors were minced and digested using Native Bacillus licheniformis protease (Creative Enzymes, NATE-0633) on ice for 30 minutes to dissociate into single cell suspension. Tumor single cell suspensions with low viability were further processed using dead cell removal kit (Miltenyi, 130-090-101). Cultured cells were collected stained with TotalSeq anti-human hashtags C0251- C0258 (Biolegend), viability dye (zombie red, Biolegend 423109) and anti-human CD45-FITC (clone HI30, Biolegend 304038) and enriched for live cells and up to 50% immune cells using BD FACS Aria II.Sample Treatment - Cells were plated and stimulated with 1000U/ml human IFNb (pbl assay science 11415), 500ng/ml poly:IC HMW (invivogen, tlrl-pic) transfected with lipofectamine 2000, 10ug/ml non-transfected poly(I:C) HMW, 2ug/ml SLR14 (acquired from Iwasaki lab), 1ug/ml ADU-S100 (invivogen, tlrl-nacda2r), 10ug/ml anti-human CD279 antibody (Biolegend, 329902), and cultured for up to 48 hours.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - Cell Ranger software was used to align the 5’ single cell sequencing reads in FASTQ files to reference GRCh38-2020-A.Data Transformation - Cell Ranger software was used to count gene expression, capture antibody hashtags and assemble V(D)J transcript sequences.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNA from single cellsHomo sapiensJeffrey IshizukafalsePerturbational sequencing of patient tumors reveals midkine as an innate immune checkpoint in neuroendocrine tumorsWe developed PERCEPT, an approach that uses ex vivo perturbational single-cell RNA sequencing to compare the response of immunomodulatory treatments with unstimulated controls directly in patient samples. Using PERCEPT, we tested cytokines and innate immune agonists in melanoma and Merkel cell carcinoma (MCC) and identified the dsRNA mimetic, RIG-I agonist, Stem Loop RNA (SLR) 14 as a powerful inducer of anti-viral states and enhancer of T cell activation. Our results demonstrate the utility of high-dimensional controlled perturbation of patient samples to identify mechanisms of innate immune response and resistance.2025-11-23T00:00:00Z2026-05-27T13:08:35.025Z2025-10-29T14:23:23.595ZE-MTAB-15917ERP183300EFO_0002944EFO_0004170EFO_0005684EFO_0004917EFO_0005518EFO_0003816EFO_0003969EFO_0004184