RESUMO
High-throughput imaging (HTI) generates complex imaging datasets from a large number of experimental perturbations. Commercial HTI software programs for image analysis workflows typically do not allow full customization and adoption of new image processing algorithms in the analysis modules. While open-source HTI analysis platforms provide individual modules in the workflow, like nuclei segmentation, spot detection, or cell tracking, they are often limited in integrating novel analysis modules or algorithms. Here, we introduce the High-Throughput Image Processing Software (HiTIPS) to expand the range and customization of existing HTI analysis capabilities. HiTIPS incorporates advanced image processing and machine learning algorithms for automated cell and nuclei segmentation, spot signal detection, nucleus tracking, nucleus registration, spot tracking, and quantification of spot signal intensity. Furthermore, HiTIPS features a graphical user interface that is open to integration of new analysis modules for existing analysis pipelines and to adding new analysis modules. To demonstrate the utility of HiTIPS, we present three examples of image analysis workflows for high-throughput DNA FISH, immunofluorescence (IF), and live-cell imaging of transcription in single cells. Altogether, we demonstrate that HiTIPS is a user-friendly, flexible, and open-source HTI software platform for a variety of cell biology applications.
Assuntos
Núcleo Celular , Processamento de Imagem Assistida por Computador , Software , Núcleo Celular/genética , Núcleo Celular/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Humanos , Algoritmos , Hibridização in Situ Fluorescente/métodos , Expressão Gênica , Aprendizado de MáquinaRESUMO
The association of genomic loci to the nuclear periphery is proposed to facilitate cell type-specific gene repression and influence cell fate decisions. However, the interplay between gene position and expression remains incompletely understood, in part because the proteins that position genomic loci at the nuclear periphery remain unidentified. Here, we used an Oligopaint-based HiDRO screen targeting â¼1000 genes to discover novel regulators of nuclear architecture in Drosophila cells. We identified the heterochromatin-associated protein Stonewall (Stwl) as a factor promoting perinuclear chromatin positioning. In female germline stem cells (GSCs), Stwl binds and positions chromatin loci, including GSC differentiation genes, at the nuclear periphery. Strikingly, Stwl-dependent perinuclear positioning is associated with transcriptional repression, highlighting a likely mechanism for Stwl's known role in GSC maintenance and ovary homeostasis. Thus, our study identifies perinuclear anchors in Drosophila and demonstrates the importance of gene repression at the nuclear periphery for cell fate.
Assuntos
Diferenciação Celular , Núcleo Celular , Cromatina , Proteínas de Drosophila , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Cromatina/metabolismo , Cromatina/genética , Núcleo Celular/metabolismo , Núcleo Celular/genética , Feminino , Diferenciação Celular/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células-Tronco/metabolismo , Células-Tronco/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Drosophila/genética , Células Germinativas/metabolismoRESUMO
The spatial arrangement of the genome within the nucleus is a pivotal aspect of cellular organization and function with implications for gene expression and regulation. While all genome organization features, such as loops, domains, and radial positioning, are nonrandom, they are characterized by a high degree of single-cell variability. Imaging approaches are ideally suited to visualize, measure, and study single-cell heterogeneity in genome organization. Here, we describe two methods for the detection of DNA and RNA of individual gene alleles by fluorescence in situ hybridization (FISH) in a high-throughput format. We have optimized combined DNA/RNA FISH approaches either using simultaneous or sequential detection of DNA and nascent RNA. These optimized DNA and RNA FISH protocols were implemented in a 384-well plate format alongside automated image and data analysis and enable accurate detection of individual gene alleles and their gene expression status across a large cell population. We successfully visualized MYC and EGFR DNA and nascent RNA with allele-level resolution in multiple cell types, and we determined the radial position of active and inactive MYC and EGFR alleles. These optimized DNA/RNA detection approaches are versatile and sensitive tools for mapping of chromatin features and gene activity at the single-allele level and at high throughput.
Assuntos
Alelos , Cromatina , Hibridização in Situ Fluorescente , Cromatina/metabolismo , Cromatina/química , Cromatina/genética , Humanos , Transcrição Gênica , Ensaios de Triagem em Larga Escala , RNA/análise , RNA/metabolismo , RNA/genética , DNA/análiseRESUMO
The spatial arrangement of the genome within the nucleus is a pivotal aspect of cellular organization and function with implications for gene expression and regulation. While all genome organization features, such as loops, domains, and radial positioning, are non-random, they are characterized by a high degree of single-cell variability. Imaging approaches are ideally suited to visualize, measure, and study single-cell heterogeneity in genome organization. Here, we describe two methods for the detection of DNA and RNA of individual gene alleles by fluorescence in situ hybridization (FISH) in a high-throughput format. We have optimized combined DNA/RNA FISH approaches either using simultaneous or sequential detection. These optimized DNA and RNA FISH protocols, implemented in a 384-well plate format alongside automated image and data analysis, enable accurate detection of chromatin loci and their gene expression status across a large cell population with allele-level resolution. We successfully visualized MYC and EGFR DNA and RNA in multiple cell types, and we determined the radial position of active and inactive MYC and EGFR alleles. These optimized DNA/RNA detection approaches are versatile and sensitive tools for mapping of chromatin features and gene activity at the single-allele level and at high throughput.
RESUMO
The spatial arrangement of the genome within the nucleus is a pivotal aspect of cellular organization and function with implications for gene expression and regulation. While all genome organization features, such as loops, domains, and radial positioning, are non-random, they are characterized by a high degree of single-cell variability. Imaging approaches are ideally suited to visualize, measure, and study single-cell heterogeneity in genome organization. Here, we describe two methods for the detection of DNA and RNA of individual gene alleles by fluorescence in situ hybridization (FISH) in a high-throughput format. We have optimized combined DNA/RNA FISH approaches either using simultaneous or sequential detection. These optimized DNA and RNA FISH protocols, implemented in a 384-well plate format alongside automated image and data analysis, enable accurate detection of chromatin loci and their gene expression status across a large cell population with allele-level resolution. We successfully visualized MYC and EGFR DNA and RNA in multiple cell types, and we determined the radial position of active and inactive MYC and EGFR alleles. These optimized DNA/RNA detection approaches are versatile and sensitive tools for mapping of chromatin features and gene activity at the single-allele level and at high throughput.
RESUMO
Functional genomics and chemical screens can identify and characterize novel cellular factors regulating signaling networks and chemical tools to modulate their function for the treatment of disease. Screening methods have relied primarily on immortalized and/or transformed cancer cell lines, which can limit the generalization of results to more physiologically relevant systems. Most have also relied on immunofluorescence, or on stably expressed recombinant fluorescent proteins, to detect specific protein markers using high-content imaging readouts. In comparison, high-throughput methods to visualize and measure RNA species have been less explored. To address this, we have adapted an isothermal signal amplification chemistry for RNA FISH known as hybridization chain reaction (HCR) to an automated, high-content imaging assay format. We present a detailed protocol for this technique, which we have named high-content HCR (hcHCR). The protocol focuses on the measurement of changes in mRNA abundance at the single-cell level in human primary cells, but it can be applied to a variety of primary cell types and perturbing agents. We anticipate that hcHCR will be most suitable for low- to medium-throughput screening experiments in which changes in transcript abundance are the desired output measure.
Assuntos
Diagnóstico por Imagem , RNA , Humanos , RNA/genética , RNA Mensageiro/genética , Hibridização de Ácido Nucleico , Transdução de SinaisRESUMO
Progesterone receptor (PR)-interacting compounds in the environment are associated with serious health hazards. However, methods for their detection in environmental samples are cumbersome. We report a sensitive activity-based biosensor for rapid and reliable screening of progesterone receptor (PR)-interacting endocrine disrupting chemicals (EDCs). The biosensor is a cell line which expresses nuclear mCherry-NF1 and a green fluorescent protein (GFP)-tagged chimera of glucocorticoid receptor (GR) N terminus fused to the ligand binding domain (LBD) of PR (GFP-GR-PR). As this LBD is shared by the PRA and PRB, the biosensor reports on the activation of both PR isoforms. This GFP-GR-PR chimera is cytoplasmic in the absence of hormone and translocates rapidly to the nucleus in response to PR agonists or antagonists in concentration- and time-dependent manner. In live cells, presence of nuclear NF1 label eliminates cell fixation and nuclear staining resulting in efficient screening. The assay can be used in screens for novel PR ligands and PR-interacting contaminants in environmental samples. A limited screen of river water samples indicated a widespread, low-level contamination with PR-interacting contaminants in all tested samples.
Assuntos
Disruptores Endócrinos , Receptores de Progesterona/genética , Bioensaio , Linhagem Celular , Citoplasma , Proteínas de Fluorescência Verde/genética , Receptores de Glucocorticoides/genéticaRESUMO
RNA-fluorescence in situ hybridization (RNA-FISH) is an essential and widely used tool for visualizing RNA molecules in intact cells. Recent advances have increased RNA-FISH sensitivity, signal detection efficiency, and throughput. However, detection of endogenous mRNA splice variants has been challenging due to the limits of visualization of RNA-FISH fluorescence signals and due to the limited number of RNA-FISH probes per target. HiFENS (high-throughput FISH detection of endogenous pre-mRNA splicing isoforms) is a method that enables visualization and relative quantification of mRNA splice variants at single-cell resolution in an automated high-throughput manner. HiFENS incorporates HCR (hybridization chain reaction) signal amplification strategies to enhance the fluorescence signal generated by low abundance transcripts or a small number of FISH probes targeting short stretches of RNA, such as single exons. The technique offers a significant advance in high-throughput FISH-based RNA detection and provides a powerful tool that can be used as a readout in functional genomics screens to discover and dissect cellular pathways regulating gene expression and alternative pre-mRNA splicing events.
Assuntos
Precursores de RNA , RNA , RNA/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Hibridização in Situ Fluorescente/métodos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Hibridização de Ácido Nucleico , Processamento AlternativoRESUMO
Alternative lengthening of telomeres (ALT) is a telomerase-independent and recombination-based mechanism used by approximately 15% of human cancers to maintain telomere length and to sustain proliferation. ALT-positive cells display unique features that could be exploited for tailored cancer therapies. A key limitation for the development of ALT-specific treatments is the lack of an assay to detect ALT-positive cells that is easy to perform and that can be scaled up. One of the most broadly used assays for ALT detection, CCA (C-circle assay), does not provide single-cell information and it is not amenable to High-Throughput Screening (HTS). To overcome these limitations, we developed Native-FISH (N-FISH) as an alternative method to visualize ALT-specific single-stranded telomeric DNA. N-FISH produces single-cell data, can be applied to fixed tissues, does not require DNA isolation or amplification steps, and it can be miniaturized in a 384-well format. This protocol details the steps to perform N-FISH protocol both in a low- and high-throughput format to analyze ALT. While low-throughput N-FISH is useful to assay the ALT state of cell lines, we expect that the miniaturized N-FISH assay coupled with high-throughput imaging will be useful in functional genomics and chemical screens to identify novel cellular factors that regulate ALT and potential ALT therapeutic targets for cancer therapies directed against ALT-positive tumors, respectively.
Assuntos
Ensaios de Triagem em Larga Escala , Neoplasias , Humanos , Animais , DNA , Telômero/genética , Peixes/genéticaRESUMO
High-throughput imaging (HTI) generates complex imaging datasets from a large number of experimental perturbations. Commercial HTI software for image analysis workflows does not allow full customization and adoption of new image processing algorithms in the analysis modules. While open-source HTI analysis platforms provide individual modules in the workflow, like nuclei segmentation, spot detection, or cell tracking, they are often limited in integrating novel analysis modules or algorithms. Here, we introduce the High-Throughput Image Processing Software (HiTIPS) to expand the range and customization of existing HTI analysis capabilities. HiTIPS incorporates advanced image processing and machine learning algorithms for automated cell and nuclei segmentation, spot signal detection, nucleus tracking, spot tracking, and quantification of spot signal intensity. Furthermore, HiTIPS features a graphical user interface that is open to integration of new algorithms for existing analysis pipelines and to adding new analysis pipelines through separate plugins. To demonstrate the utility of HiTIPS, we present three examples of image analysis workflows for high-throughput DNA FISH, immunofluorescence (IF), and live-cell imaging of transcription in single cells. Altogether, we demonstrate that HiTIPS is a user-friendly, flexible, and open-source HTI analysis platform for a variety of cell biology applications.
RESUMO
The association of genomic loci to the nuclear periphery is proposed to facilitate cell-type specific gene repression and influence cell fate decisions. However, the interplay between gene position and expression remains incompletely understood, in part because the proteins that position genomic loci at the nuclear periphery remain unidentified. Here, we used an Oligopaint-based HiDRO screen targeting ~1000 genes to discover novel regulators of nuclear architecture in Drosophila cells. We identified the heterochromatin-associated protein, Stonewall (Stwl), as a factor promoting perinuclear chromatin positioning. In female germline stem cells (GSCs), Stwl binds and positions chromatin loci, including GSC differentiation genes, at the nuclear periphery. Strikingly, Stwl-dependent perinuclear positioning is associated with transcriptional repression, highlighting a likely mechanism for Stwl's known role in GSC maintenance and ovary homeostasis. Thus, our study identifies perinuclear anchors in Drosophila and demonstrates the importance of gene repression at the nuclear periphery for cell fate.
RESUMO
The shape and size of the human cell nucleus is highly variable among cell types and tissues. Changes in nuclear morphology are associated with disease, including cancer, as well as with premature and normal aging. Despite the very fundamental nature of nuclear morphology, the cellular factors that determine nuclear shape and size are not well understood. To identify regulators of nuclear architecture in a systematic and unbiased fashion, we performed a high-throughput imaging-based siRNA screen targeting 867 nuclear proteins including chromatin-associated proteins, epigenetic regulators, and nuclear envelope components. Using multiple morphometric parameters, and eliminating cell cycle effectors, we identified a set of novel determinants of nuclear size and shape. Interestingly, most identified factors altered nuclear morphology without affecting the levels of lamin proteins, which are known prominent regulators of nuclear shape. In contrast, a major group of nuclear shape regulators were modifiers of repressive heterochromatin. Biochemical and molecular analysis uncovered a direct physical interaction of histone H3 with lamin A mediated via combinatorial histone modifications. Furthermore, disease-causing lamin A mutations that result in disruption of nuclear shape inhibited lamin A-histone H3 interactions. Oncogenic histone H3.3 mutants defective for H3K27 methylation resulted in nuclear morphology abnormalities. Altogether, our results represent a systematic exploration of cellular factors involved in determining nuclear morphology and they identify the interaction of lamin A with histone H3 as an important contributor to nuclear morphology in human cells.
Assuntos
Histonas , Lamina Tipo A , Humanos , Histonas/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Membrana Nuclear/metabolismo , Epigênese GenéticaRESUMO
One of the major cellular mechanisms to ensure cellular protein homeostasis is the endoplasmic reticulum (ER) stress response. This pathway is triggered by accumulation of misfolded proteins in the ER lumen. The ER stress response is also activated in the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). Here, we explore the mechanism of activation of the ER stress response in HGPS. We find that aggregation of the diseases-causing progerin protein at the nuclear envelope triggers ER stress. Induction of ER stress is dependent on the inner nuclear membrane protein SUN2 and its ability to cluster in the nuclear membrane. Our observations suggest that the presence of nucleoplasmic protein aggregates can be sensed, and signaled to the ER lumen, via clustering of SUN2. These results identify a mechanism of communication between the nucleus and the ER and provide insight into the molecular disease mechanisms of HGPS.
Assuntos
Senilidade Prematura , Progéria , Humanos , Senilidade Prematura/metabolismo , Membrana Nuclear/metabolismo , Núcleo Celular/metabolismo , Progéria/metabolismo , Proteínas de Membrana/metabolismo , Estresse do Retículo Endoplasmático , Lamina Tipo A/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismoRESUMO
Restricting the localization of the evolutionarily conserved centromeric histone H3 variant CENP-A to centromeres prevents chromosomal instability (CIN). The mislocalization of CENP-A to non-centromeric regions contributes to CIN in yeasts, flies and human cells. Even though overexpression and mislocalization of CENP-A have been reported in cancers, the mechanisms responsible for its mislocalization remain poorly understood. Here, we used an imaging-based high-throughput RNAi screen to identify factors that prevent mislocalization of overexpressed YFP-tagged CENP-A (YFP-CENP-A) in HeLa cells. Among the top five candidates in the screen - the depletion of which showed increased nuclear YFP-CENP-A fluorescence - were the histone chaperones CHAF1B (or p60) and CHAF1A (or p150). Follow-up validation and characterization experiments showed that CHAF1B-depleted cells exhibited CENP-A mislocalization, CIN phenotypes and increased enrichment of CENP-A in chromatin fractions. The depletion of DAXX, a histone H3.3 chaperone, suppressed CENP-A mislocalization and CIN in CHAF1B-depleted cells. We propose that in CHAF1B-depleted cells, DAXX promotes mislocalization of the overexpressed CENP-A to non-centromeric regions, resulting in CIN. In summary, we identified regulators of CENP-A localization and defined a role for CHAF1B in preventing DAXX-dependent CENP-A mislocalization and CIN.
Assuntos
Proteínas Cromossômicas não Histona , Histonas , Humanos , Histonas/genética , Proteína Centromérica A/genética , Células HeLa , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromatina , Centrômero/metabolismo , Chaperonas Moleculares/metabolismo , Instabilidade Cromossômica , Autoantígenos/genética , Fator 1 de Modelagem da Cromatina/genéticaRESUMO
Human Histone Locus Bodies (HLBs) are nuclear subdomains comprised of clustered histone genes that are coordinately regulated throughout the cell cycle. We addressed temporal-spatial higher-order genome organization for time-dependent chromatin remodeling at HLBs that supports control of cell proliferation. Proximity distances of specific genomic contacts within histone gene clusters exhibit subtle changes during the G1 phase in MCF10 breast cancer progression model cell lines. This approach directly demonstrates that the two principal histone gene regulatory proteins, HINFP (H4 gene regulator) and NPAT, localize at chromatin loop anchor-points, denoted by CTCF binding, supporting the stringent requirement for histone biosynthesis to package newly replicated DNA as chromatin. We identified a novel enhancer region located â¼ 2 MB distal to histone gene sub-clusters on chromosome 6 that consistently makes genomic contacts with HLB chromatin and is bound by NPAT. During G1 progression the first DNA loops form between one of three histone gene sub-clusters bound by HINFP and the distal enhancer region. Our findings are consistent with a model that the HINFP/NPAT complex controls the formation and dynamic remodeling of higher-order genomic organization of histone gene clusters at HLBs in early to late G1 phase to support transcription of histone mRNAs in S phase.
Assuntos
Neoplasias da Mama , Histonas , Humanos , Feminino , Histonas/genética , Histonas/metabolismo , Cromatina/genética , Neoplasias da Mama/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Corpos Nucleares , Família MultigênicaRESUMO
Splicing factors play an essential role in regulation of alternative pre-mRNA splicing. While much progress has been made in delineating the mechanisms of the splicing machinery, the identity of signal transduction pathways and upstream factors that regulate splicing factor activity is largely unknown. A major challenge in the discovery of upstream regulatory factors of pre-mRNA splicing is the scarcity of functional genomics screening methods to monitor splicing outcomes of endogenous genes. Here, we have developed HiFENS (high throughput FISH detection of endogenous splicing isoforms), a high-throughput imaging assay based on hybridization chain reaction (HCR) and used HiFENS to screen for cellular factors that regulate alternative splicing of endogenous genes. We demonstrate optimized detection with high specificity of endogenous splicing isoforms and multiplexing of probes for accurate detection of splicing outcomes with single cell resolution. As proof-of-principle, we perform an RNAi screen of 702 human kinases and identify potential candidate upstream splicing regulators of the FGFR2 gene. HiFENS should be a useful tool for the unbiased delineation of cellular pathways involved in alternative splicing regulation.
Assuntos
Processamento Alternativo , Hibridização in Situ Fluorescente , Precursores de RNA , Humanos , Éxons , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , Precursores de RNA/genética , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/metabolismo , Hibridização in Situ Fluorescente/métodosRESUMO
Chromatin insulators are DNA-protein complexes that can prevent the spread of repressive chromatin and block communication between enhancers and promoters to regulate gene expression. In Drosophila, the gypsy chromatin insulator complex consists of three core proteins: CP190, Su(Hw), and Mod(mdg4)67.2. These factors concentrate at nuclear foci termed insulator bodies, and changes in insulator body localization have been observed in mutants defective for insulator function. Here, we identified NURF301/E(bx), a nucleosome remodeling factor, as a novel regulator of gypsy insulator body localization through a high-throughput RNAi imaging screen. NURF301 promotes gypsy-dependent insulator barrier activity and physically interacts with gypsy insulator proteins. Using ChIP-seq, we found that NURF301 co-localizes with insulator proteins genome-wide, and NURF301 promotes chromatin association of Su(Hw) and CP190 at gypsy insulator binding sites. These effects correlate with NURF301-dependent nucleosome repositioning. At the same time, CP190 and Su(Hw) both facilitate recruitment of NURF301 to chromatin. Finally, Oligopaint FISH combined with immunofluorescence revealed that NURF301 promotes 3D contact between insulator bodies and gypsy insulator DNA binding sites, and NURF301 is required for proper nuclear positioning of gypsy binding sites. Our data provide new insights into how a nucleosome remodeling factor and insulator proteins cooperatively contribute to nuclear organization.
Assuntos
Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Animais , Cromatina/genética , Cromatina/metabolismo , DNA/metabolismo , Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Elementos Isolantes/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismoRESUMO
High-throughput DNA fluorescence in situ hybridization (hiFISH) combines multicolor combinatorial DNA FISH staining with automated image acquisition and analysis to visualize and localize tens to hundreds of genomic loci in up to millions of cells. hiFISH can be used to measure physical distances between pairs of genomic loci, radial distances from genomic loci to the nuclear edge or center, and distances between genomic loci and nuclear structures defined by protein or RNA markers. The resulting large datasets of 3D spatial distances can be used to study cellular heterogeneity in genome architecture and the molecular mechanisms underlying this phenomenon in a variety of cellular systems. In this chapter we provide detailed protocols for hiFISH to measure distances between genomic loci, including all steps involved in DNA FISH probe design and preparation, cell culture, DNA FISH staining in 384-well imaging plates, automated image acquisition and analysis, and, finally, statistical analysis.
Assuntos
Núcleo Celular , DNA , Núcleo Celular/metabolismo , DNA/química , Sondas de DNA/metabolismo , Genoma , Hibridização in Situ Fluorescente/métodosRESUMO
Measurement of gene expression at the single-cell level has advanced the study of transcriptional regulation programs in healthy and disease states. In particular, single-cell approaches have shed light on the high level of transcriptional heterogeneity of individual cells, both at baseline and in response to experimental or environmental perturbations. We have developed a method for high-content imaging (HCI)-based quantification of relative changes in transcript abundance at the single-cell level in human primary immune cells and have validated its performance under multiple experimental conditions to demonstrate its general applicability. This method, named hcHCR, combines the sensitivity of the hybridization chain reaction (HCR) for the visualization of RNA in single cells, with the speed, scalability, and reproducibility of HCI. We first tested eight cell attachment substrates for short-term culture of primary human B cells, T cells, monocytes, or neutrophils. We then miniaturized HCR in 384-well format and documented the ability of the method to detect changes in transcript abundance at the single-cell level in thousands of cells for each experimental condition by HCI. Furthermore, we demonstrated the feasibility of multiplexing gene expression measurements by simultaneously assaying the abundance of three transcripts per cell at baseline and in response to an experimental stimulus. Finally, we tested the robustness of the assay to technical and biological variation. We anticipate that hcHCR will be suitable for low- to medium-throughput chemical or functional genomics screens in primary human cells, with the possibility of performing screens on cells obtained from patients with a specific disease.