RESUMO
To better understand host-virus genetic dependencies and find potential therapeutic targets for COVID-19, we performed a genome-scale CRISPR loss-of-function screen to identify host factors required for SARS-CoV-2 viral infection of human alveolar epithelial cells. Top-ranked genes cluster into distinct pathways, including the vacuolar ATPase proton pump, Retromer, and Commander complexes. We validate these gene targets using several orthogonal methods such as CRISPR knockout, RNA interference knockdown, and small-molecule inhibitors. Using single-cell RNA-sequencing, we identify shared transcriptional changes in cholesterol biosynthesis upon loss of top-ranked genes. In addition, given the key role of the ACE2 receptor in the early stages of viral entry, we show that loss of RAB7A reduces viral entry by sequestering the ACE2 receptor inside cells. Overall, this work provides a genome-scale, quantitative resource of the impact of the loss of each host gene on fitness/response to viral infection.
Assuntos
COVID-19/genética , COVID-19/virologia , Interações Hospedeiro-Patógeno , SARS-CoV-2/fisiologia , Células A549 , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , Enzima de Conversão de Angiotensina 2/metabolismo , Vias Biossintéticas , COVID-19/metabolismo , Colesterol/biossíntese , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endossomos/metabolismo , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes/métodos , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Interferência de RNA , SARS-CoV-2/crescimento & desenvolvimento , Análise de Célula Única , Carga Viral/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/genética , proteínas de unión al GTP Rab7RESUMO
The simultaneous measurement of multiple modalities represents an exciting frontier for single-cell genomics and necessitates computational methods that can define cellular states based on multimodal data. Here, we introduce "weighted-nearest neighbor" analysis, an unsupervised framework to learn the relative utility of each data type in each cell, enabling an integrative analysis of multiple modalities. We apply our procedure to a CITE-seq dataset of 211,000 human peripheral blood mononuclear cells (PBMCs) with panels extending to 228 antibodies to construct a multimodal reference atlas of the circulating immune system. Multimodal analysis substantially improves our ability to resolve cell states, allowing us to identify and validate previously unreported lymphoid subpopulations. Moreover, we demonstrate how to leverage this reference to rapidly map new datasets and to interpret immune responses to vaccination and coronavirus disease 2019 (COVID-19). Our approach represents a broadly applicable strategy to analyze single-cell multimodal datasets and to look beyond the transcriptome toward a unified and multimodal definition of cellular identity.
Assuntos
SARS-CoV-2/imunologia , Análise de Célula Única/métodos , Células 3T3 , Animais , COVID-19/imunologia , Linhagem Celular , Perfilação da Expressão Gênica/métodos , Humanos , Imunidade/imunologia , Leucócitos Mononucleares/imunologia , Linfócitos/imunologia , Camundongos , Análise de Sequência de RNA/métodos , Transcriptoma/imunologia , VacinaçãoRESUMO
In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.
Assuntos
Cromatina , Epigênese Genética , Genótipo , Mutação , Análise de Célula Única , Animais , Feminino , Humanos , Masculino , Camundongos , Antígenos CD34/metabolismo , Diferenciação Celular/genética , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética/genética , Epigenoma/genética , Genoma Mitocondrial/genética , Técnicas de Genotipagem , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Inflamação/genética , Inflamação/patologia , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Megacariócitos/metabolismo , Megacariócitos/patologia , Proteínas de Membrana/genética , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Transtornos Mieloproliferativos/patologia , RNA/genética , Células Clonais/metabolismoRESUMO
The engineering of autologous patient T cells for adoptive cell therapies has revolutionized the treatment of several types of cancer1. However, further improvements are needed to increase response and cure rates. CRISPR-based loss-of-function screens have been limited to negative regulators of T cell functions2-4 and raise safety concerns owing to the permanent modification of the genome. Here we identify positive regulators of T cell functions through overexpression of around 12,000 barcoded human open reading frames (ORFs). The top-ranked genes increased the proliferation and activation of primary human CD4+ and CD8+ T cells and their secretion of key cytokines such as interleukin-2 and interferon-γ. In addition, we developed the single-cell genomics method OverCITE-seq for high-throughput quantification of the transcriptome and surface antigens in ORF-engineered T cells. The top-ranked ORF-lymphotoxin-ß receptor (LTBR)-is typically expressed in myeloid cells but absent in lymphocytes. When overexpressed in T cells, LTBR induced profound transcriptional and epigenomic remodelling, leading to increased T cell effector functions and resistance to exhaustion in chronic stimulation settings through constitutive activation of the canonical NF-κB pathway. LTBR and other highly ranked genes improved the antigen-specific responses of chimeric antigen receptor T cells and γδ T cells, highlighting their potential for future cancer-agnostic therapies5. Our results provide several strategies for improving next-generation T cell therapies by the induction of synthetic cell programmes.
Assuntos
Linfócitos T CD8-Positivos , Neoplasias , Linfócitos T CD4-Positivos , Proliferação de Células , Humanos , Imunoterapia Adotiva , Ativação Linfocitária/genéticaRESUMO
Pooled CRISPR screens coupled with single-cell RNA-sequencing have enabled systematic interrogation of gene function and regulatory networks. Here, we introduce Cas13 RNA Perturb-seq (CaRPool-seq), which leverages the RNA-targeting CRISPR-Cas13d system and enables efficient combinatorial perturbations alongside multimodal single-cell profiling. CaRPool-seq encodes multiple perturbations on a cleavable CRISPR array that is associated with a detectable barcode sequence, allowing for the simultaneous targeting of multiple genes. We compared CaRPool-seq to existing Cas9-based methods, highlighting its unique strength to efficiently profile combinatorially perturbed cells. Finally, we apply CaRPool-seq to perform multiplexed combinatorial perturbations of myeloid differentiation regulators in an acute myeloid leukemia (AML) model system and identify extensive interactions between different chromatin regulators that can enhance or suppress AML differentiation phenotypes.
Assuntos
Cromatina , RNA , RNA/genética , Sistemas CRISPR-Cas/genéticaRESUMO
Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of mature T-cell neoplasms characterized by the accumulation of clonal malignant CD4+ T cells in the skin. The most common variant of CTCL, mycosis fungoides (MF ), is confined to the skin in early stages but can be accompanied by extracutaneous dissemination of malignant T cells to the blood and lymph nodes in advanced stages of disease. Sézary syndrome (SS), a leukemic form of disease, is characterized by significant blood involvement. Little is known about the transcriptional and genomic relationship between skin- and blood-residing malignant T cells in CTCL. To identify and interrogate malignant clones in matched skin and blood from patients with leukemic MF and SS, we combine T-cell receptor clonotyping with quantification of gene expression and cell surface markers at the single cell level. Our data reveal clonal evolution at a transcriptional and genetic level within the malignant populations of individual patients. We highlight highly consistent transcriptional signatures delineating skin- and blood-derived malignant T cells. Analysis of these 2 populations suggests that environmental cues, along with genetic aberrations, contribute to transcriptional profiles of malignant T cells. Our findings indicate that the skin microenvironment in CTCL promotes a transcriptional response supporting rapid malignant expansion, as opposed to the quiescent state observed in the blood, potentially influencing efficacy of therapies. These results provide insight into tissue-specific characteristics of cancerous cells and underscore the need to address the patients' individual malignant profiles at the time of therapy to eliminate all subclones.
Assuntos
Linfoma Cutâneo de Células T/patologia , Neoplasias Cutâneas/patologia , Células Cultivadas , Humanos , Linfoma Cutâneo de Células T/genética , Análise de Célula Única , Neoplasias Cutâneas/genética , Transcriptoma , Células Tumorais CultivadasRESUMO
Multimodal single-cell assays provide high-resolution snapshots of complex cell populations, but are mostly limited to transcriptome plus an additional modality. Here, we describe expanded CRISPR-compatible cellular indexing of transcriptomes and epitopes by sequencing (ECCITE-seq) for the high-throughput characterization of at least five modalities of information from each single cell. We demonstrate application of ECCITE-seq to multimodal CRISPR screens with robust direct single-guide RNA capture and to clonotype-aware multimodal phenotyping of cancer samples.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteínas/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcriptoma/genética , Animais , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Perfilação da Expressão Gênica , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Linfoma Cutâneo de Células T/genética , Linfoma Cutâneo de Células T/metabolismo , Linfoma Cutâneo de Células T/patologia , Camundongos , Células NIH 3T3 , RNA Guia de Cinetoplastídeos/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Células Tumorais CultivadasRESUMO
In this study, we investigate the interplay between Ku, a central non-homologous end-joining component, and the Mre11-Rad50-Xrs2 (MRX) complex and Sae2, end-processing factors crucial for initiating 5'-3' resection of double-strand break (DSB) ends. We show that in the absence of end protection by Ku, the requirement for the MRX complex is bypassed and resection is executed by Exo1. In contrast, both the Exo1 and Sgs1 resection pathways contribute to DSB processing in the absence of Ku and Sae2 or when the MRX complex is intact, but functionally compromised by elimination of the Mre11 nuclease activity. The ionizing radiation sensitivity of a mutant defective for extensive resection (exo1Δ sgs1Δ) cannot be suppressed by the yku70Δ mutation, indicating that Ku suppression is specific to the initiation of resection. We provide evidence that replication-associated DSBs need to be processed by Sae2 for repair by homologous recombination unless Ku is absent. Finally, we show that the presence of Ku exacerbates DNA end-processing defects established in the sae2Δ sgs1Δ mutant, leading to its lethality.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Complexos Multiproteicos/metabolismo , RecQ Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Southern Blotting , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Raios gama , Saccharomyces cerevisiae , Telômero/genéticaRESUMO
DNA ends exposed after introduction of double-strand breaks (DSBs) undergo 5'-3' nucleolytic degradation to generate single-stranded DNA, the substrate for binding by the Rad51 protein to initiate homologous recombination. This process is poorly understood in eukaryotes, but several factors have been implicated, including the Mre11 complex (Mre11-Rad50-Xrs2/NBS1), Sae2/CtIP/Ctp1 and Exo1. Here we demonstrate that yeast Exo1 nuclease and Sgs1 helicase function in alternative pathways for DSB processing. Novel, partially resected intermediates accumulate in a double mutant lacking Exo1 and Sgs1, which are poor substrates for homologous recombination. The early processing step that generates partly resected intermediates is dependent on Sae2. When Sae2 is absent, in addition to Exo1 and Sgs1, unprocessed DSBs accumulate and homology-dependent repair fails. These results suggest a two-step mechanism for DSB processing during homologous recombination. First, the Mre11 complex and Sae2 remove a small oligonucleotide(s) from the DNA ends to form an early intermediate. Second, Exo1 and/or Sgs1 rapidly process this intermediate to generate extensive tracts of single-stranded DNA that serve as substrate for Rad51.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Exodesoxirribonucleases/metabolismo , RecQ Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases , Exodesoxirribonucleases/genética , Modelos Biológicos , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , RecQ Helicases/genética , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Homologous recombination (HR)-mediated DNA double-strand break repair maintains genome integrity. Although long-studied, an understanding of two essential steps in this process -- the resection of DNA ends to produce recombinogenic 3' single-stranded DNA tails and the resolution of recombination intermediates -- has remained elusive. Recent findings show an unexpected role for the Sgs1 (BLM) helicase and Dna2 nuclease in end resection, and provide mechanistic insight into the initiation of 5'-3' resection as well as its regulation by the cell cycle and the DNA damage response. Moreover, the identification of a novel Holliday junction resolvase, Yen1 (GEN1), and several helicases that dismantle strand invasion intermediates has increased the repertoire of nucleases and helicases capable of resolving recombination intermediates.
Assuntos
DNA Helicases/fisiologia , Recombinação Genética/genética , Animais , DNA Helicases/metabolismo , Reparo do DNA/genética , Reparo do DNA/fisiologia , Resolvases de Junção Holliday/metabolismo , Resolvases de Junção Holliday/fisiologia , Humanos , Modelos Biológicos , Recombinação Genética/fisiologiaRESUMO
DNA double-strand breaks (DSBs) are nucleolytically processed to generate single-stranded DNA tails for homologous recombination. In Saccharomyces cerevisiae meiosis, this 5'-to-3' resection involves initial nicking by the Mre11-Rad50-Xrs2 complex (MRX) plus Sae2, then exonucleolytic digestion by Exo1. Chromatin remodeling adjacent to meiotic DSBs is thought to be necessary for resection, but the relevant remodeling activity was unknown. Here we show that the SWI/SNF-like ATPase Fun30 plays a major, non-redundant role in resecting meiotic DSBs. A fun30 null mutation shortened resection tract lengths almost as severely as an exo1-nd (nuclease-dead) mutation, and resection was further shortened in the fun30 exo1-nd double mutant. Fun30 associates with chromatin in response to meiotic DSBs, and the constitutive positioning of nucleosomes governs resection endpoint locations in the absence of Fun30. We infer that Fun30 directly promotes both the MRX- and Exo1-dependent steps in resection, possibly by removing nucleosomes from broken chromatids. Moreover, we found that the extremely short resection in the fun30 exo1-nd double mutant is accompanied by compromised interhomolog recombination bias, leading to defects in recombination and chromosome segregation. Thus, this study also provides insight about the minimal resection lengths needed for robust recombination.
RESUMO
While methods such as the Assay for Transposase Accessible Chromatin by sequencing (ATAC-seq) enable a comprehensive characterization of regulatory DNA, additional measurements are required to characterize the multifaceted nature of eukaryotic cells. Here, we delineate the ATAC with Select Antigen Profiling by sequencing (ASAP-seq) protocol, a scalable approach to quantifying proteins via oligo-tagged antibodies alongside accessible DNA in thousands of single cells. Critically, our method utilizes a custom bridge oligo that enables the utilization of a variety of oligo-conjugated antibodies, enabling the utilization and repurposing of other commercial products. The ASAP-seq method can be completed with straightforward experimental and computational modifications existing single-cell ATAC-seq workflows but yields distinct modalities underlying complex cellular states, including estimation of protein abundance on the cell surface as well as intracellular and intranuclear factors.
Assuntos
Cromatina , Sequenciamento de Nucleotídeos em Larga Escala , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , DNA/genética , Sequenciamento de Cromatina por ImunoprecipitaçãoRESUMO
Most variants associated with complex traits and diseases identified by genome-wide association studies (GWAS) map to noncoding regions of the genome with unknown effects. Using ancestrally diverse, biobank-scale GWAS data, massively parallel CRISPR screens, and single-cell transcriptomic and proteomic sequencing, we discovered 124 cis-target genes of 91 noncoding blood trait GWAS loci. Using precise variant insertion through base editing, we connected specific variants with gene expression changes. We also identified trans-effect networks of noncoding loci when cis target genes encoded transcription factors or microRNAs. Networks were themselves enriched for GWAS variants and demonstrated polygenic contributions to complex traits. This platform enables massively parallel characterization of the target genes and mechanisms of human noncoding variants in both cis and trans.
Assuntos
Doença , Estudo de Associação Genômica Ampla , Herança Multifatorial , Locos de Características Quantitativas , Análise de Célula Única , Humanos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Proteômica , Células Sanguíneas , RNA-Seq , Doença/genéticaRESUMO
SARS-CoV-2 infection and vaccination elicit potent immune responses. Our study presents a comprehensive multimodal single-cell dataset of peripheral blood of patients with acute COVID-19 and of healthy volunteers before and after receiving the SARS-CoV-2 mRNA vaccine and booster. We compared host immune responses to the virus and vaccine using transcriptional profiling, coupled with B/T cell receptor repertoire reconstruction. COVID-19 patients displayed an enhanced interferon signature and cytotoxic gene upregulation, absent in vaccine recipients. These findings were validated in an independent dataset. Analysis of B and T cell repertoires revealed that, while the majority of clonal lymphocytes in COVID-19 patients were effector cells, clonal expansion was more evident among circulating memory cells in vaccine recipients. Furthermore, while clonal αß T cell responses were observed in both COVID-19 patients and vaccine recipients, dramatic expansion of clonal γδT cells was found only in infected individuals. Our dataset enables comparative analyses of immune responses to infection versus vaccination, including clonal B and T cell responses. Integrating our data with publicly available datasets allowed us to validate our findings in larger cohorts. To our knowledge, this is the first dataset to include comprehensive profiling of longitudinal samples from healthy volunteers pre/post SARS-CoV-2 vaccine and booster.
RESUMO
SARS-CoV-2 infection and vaccination elicit potent immune responses. Our study presents a comprehensive multimodal single-cell analysis of blood from COVID-19 patients and healthy volunteers receiving the SARS-CoV-2 vaccine and booster. We profiled immune responses via transcriptional analysis and lymphocyte repertoire reconstruction. COVID-19 patients displayed an enhanced interferon signature and cytotoxic gene upregulation, absent in vaccine recipients. B and T cell repertoire analysis revealed clonal expansion among effector cells in COVID-19 patients and memory cells in vaccine recipients. Furthermore, while clonal αß T cell responses were observed in both COVID-19 patients and vaccine recipients, expansion of clonal γδ T cells was found only in infected individuals. Our dataset enables side-by-side comparison of immune responses to infection versus vaccination, including clonal B and T cell responses. Our comparative analysis shows that vaccination induces a robust, durable clonal B and T cell responses, without the severe inflammation associated with infection.
RESUMO
Pathogenic mutations in mitochondrial DNA (mtDNA) compromise cellular metabolism, contributing to cellular heterogeneity and disease. Diverse mutations are associated with diverse clinical phenotypes, suggesting distinct organ- and cell-type-specific metabolic vulnerabilities. Here we establish a multi-omics approach to quantify deletions in mtDNA alongside cell state features in single cells derived from six patients across the phenotypic spectrum of single large-scale mtDNA deletions (SLSMDs). By profiling 206,663 cells, we reveal the dynamics of pathogenic mtDNA deletion heteroplasmy consistent with purifying selection and distinct metabolic vulnerabilities across T-cell states in vivo and validate these observations in vitro. By extending analyses to hematopoietic and erythroid progenitors, we reveal mtDNA dynamics and cell-type-specific gene regulatory adaptations, demonstrating the context-dependence of perturbing mitochondrial genomic integrity. Collectively, we report pathogenic mtDNA heteroplasmy dynamics of individual blood and immune cells across lineages, demonstrating the power of single-cell multi-omics for revealing fundamental properties of mitochondrial genetics.
Assuntos
DNA Mitocondrial , Doenças Mitocondriais , Humanos , DNA Mitocondrial/genética , Multiômica , Doenças Mitocondriais/genética , Mitocôndrias/genética , MutaçãoRESUMO
Technologies that profile chromatin modifications at single-cell resolution offer enormous promise for functional genomic characterization, but the sparsity of the measurements and integrating multiple binding maps represent substantial challenges. Here we introduce single-cell (sc)CUT&Tag-pro, a multimodal assay for profiling protein-DNA interactions coupled with the abundance of surface proteins in single cells. In addition, we introduce single-cell ChromHMM, which integrates data from multiple experiments to infer and annotate chromatin states based on combinatorial histone modification patterns. We apply these tools to perform an integrated analysis across nine different molecular modalities in circulating human immune cells. We demonstrate how these two approaches can characterize dynamic changes in the function of individual genomic elements across both discrete cell states and continuous developmental trajectories, nominate associated motifs and regulators that establish chromatin states and identify extensive and cell-type-specific regulatory priming. Finally, we demonstrate how our integrated reference can serve as a scaffold to map and improve the interpretation of additional scCUT&Tag datasets.
Assuntos
Cromatina , Histonas , Cromatina/genética , Imunoprecipitação da Cromatina , DNA , Genômica , Histonas/genética , Histonas/metabolismo , HumanosRESUMO
Spo11, which makes DNA double-strand breaks (DSBs) that are essential for meiotic recombination, has long been recalcitrant to biochemical study. We provide molecular analysis of Saccharomyces cerevisiae Spo11 purified with partners Rec102, Rec104 and Ski8. Rec102 and Rec104 jointly resemble the B subunit of archaeal topoisomerase VI, with Rec104 occupying a position similar to the Top6B GHKL-type ATPase domain. Unexpectedly, the Spo11 complex is monomeric (1:1:1:1 stoichiometry), consistent with dimerization controlling DSB formation. Reconstitution of DNA binding reveals topoisomerase-like preferences for duplex-duplex junctions and bent DNA. Spo11 also binds noncovalently but with high affinity to DNA ends mimicking cleavage products, suggesting a mechanism to cap DSB ends. Mutations that reduce DNA binding in vitro attenuate DSB formation, alter DSB processing and reshape the DSB landscape in vivo. Our data reveal structural and functional similarities between the Spo11 core complex and Topo VI, but also highlight differences reflecting their distinct biological roles.
Assuntos
Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/metabolismo , Meiose/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Arqueais/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Microscopia de Força Atômica , Mutação/genética , Conformação de Ácido Nucleico , Recombinases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Simultaneous measurement of surface proteins and gene expression within single cells using oligo-conjugated antibodies offers high-resolution snapshots of complex cell populations. Signal from oligo-conjugated antibodies is quantified by high-throughput sequencing and is highly scalable and sensitive. We investigated the response of oligo-conjugated antibodies towards four variables: concentration, staining volume, cell number at staining, and tissue. We find that staining with recommended antibody concentrations causes unnecessarily high background and amount of antibody used can be drastically reduced without loss of biological information. Reducing staining volume only affects antibodies targeting abundant epitopes used at low concentrations and is counteracted by reducing cell numbers. Adjusting concentrations increases signal, lowers background, and reduces costs. Background signal can account for a major fraction of total sequencing and is primarily derived from antibodies used at high concentrations. This study provides new insight into titration response and background of oligo-conjugated antibodies and offers concrete guidelines to improve such panels.