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Natural and induced somatic mutations that accumulate in the genome during development record the phylogenetic relationships of cells; whether these lineage barcodes capture the complex dynamics of progenitor states remains unclear. We introduce quantitative fate mapping, an approach to reconstruct the hierarchy, commitment times, population sizes, and commitment biases of intermediate progenitor states during development based on a time-scaled phylogeny of their descendants. To reconstruct time-scaled phylogenies from lineage barcodes, we introduce Phylotime, a scalable maximum likelihood clustering approach based on a general barcoding mutagenesis model. We validate these approaches using realistic in silico and in vitro barcoding experiments. We further establish criteria for the number of cells that must be analyzed for robust quantitative fate mapping and a progenitor state coverage statistic to assess the robustness. This work demonstrates how lineage barcodes, natural or synthetic, enable analyzing progenitor fate and dynamics long after embryonic development in any organism.
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Desarrollo Embrionario , Linaje de la Célula/genética , Estudios Retrospectivos , Filogenia , MutagénesisRESUMEN
The four-dimensional nucleome (4DN) consortium studies the architecture of the genome and the nucleus in space and time. We summarize progress by the consortium and highlight the development of technologies for (1) mapping genome folding and identifying roles of nuclear components and bodies, proteins, and RNA, (2) characterizing nuclear organization with time or single-cell resolution, and (3) imaging of nuclear organization. With these tools, the consortium has provided over 2,000 public datasets. Integrative computational models based on these data are starting to reveal connections between genome structure and function. We then present a forward-looking perspective and outline current aims to (1) delineate dynamics of nuclear architecture at different timescales, from minutes to weeks as cells differentiate, in populations and in single cells, (2) characterize cis-determinants and trans-modulators of genome organization, (3) test functional consequences of changes in cis- and trans-regulators, and (4) develop predictive models of genome structure and function.
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Núcleo Celular , Genoma , Genoma/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismoRESUMEN
Employing DNA as a high-density data storage medium has paved the way for next-generation digital storage and biosensing technologies. However, the multipart architecture of current DNA-based recording techniques renders them inherently slow and incapable of recording fluctuating signals with subhour frequencies. To address this limitation, we developed a simplified system employing a single enzyme, terminal deoxynucleotidyl transferase (TdT), to transduce environmental signals into DNA. TdT adds nucleotides to the 3'-ends of single-stranded DNA (ssDNA) in a template-independent manner, selecting bases according to inherent preferences and environmental conditions. By characterizing TdT nucleotide selectivity under different conditions, we show that TdT can encode various physiologically relevant signals such as Co2+, Ca2+, and Zn2+ concentrations and temperature changes in vitro. Further, by considering the average rate of nucleotide incorporation, we show that the resulting ssDNA functions as a molecular ticker tape. With this method we accurately encode a temporal record of fluctuations in Co2+ concentration to within 1 min over a 60 min period. Finally, we engineer TdT to allosterically turn off in the presence of a physiologically relevant concentration of calcium. We use this engineered TdT in concert with a reference TdT to develop a two-polymerase system capable of recording a single-step change in the Ca2+ signal to within 1 min over a 60 min period. This work expands the repertoire of DNA-based recording techniques by developing a novel DNA synthesis-based system that can record temporal environmental signals into DNA with a resolution of minutes.
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ADN NucleotidilexotransferasaRESUMEN
We present an approach for engineering evolving DNA barcodes in living cells. A homing guide RNA (hgRNA) scaffold directs the Cas9-hgRNA complex to the DNA locus of the hgRNA itself. We show that this homing CRISPR-Cas9 system acts as an expressed genetic barcode that diversifies its sequence and that the rate of diversification can be controlled in cultured cells. We further evaluate these barcodes in cell populations and show that they can be used to record lineage history and that the barcode RNA can be amplified in situ, a prerequisite for in situ sequencing. This integrated approach will have wide-ranging applications, such as in deep lineage tracing, cellular barcoding, molecular recording, dissecting cancer biology, and connectome mapping.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Endonucleasas/genética , Endonucleasas/metabolismo , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Lentivirus/genética , ARN Guía de KinetoplastidaRESUMEN
Forkhead Box (Fox) proteins share the Forkhead domain, a winged-helix DNA binding module, which is conserved among eukaryotes from yeast to humans. These sequence-specific DNA binding proteins have been primarily characterized as transcription factors regulating diverse cellular processes from cell cycle control to developmental fate, deregulation of which contributes to developmental defects, cancer, and aging. We recently identified Saccharomyces cerevisiae Forkhead 1 (Fkh1) and Forkhead 2 (Fkh2) as required for the clustering of a subset of replication origins in G1 phase and for the early initiation of these origins in the ensuing S phase, suggesting a mechanistic role linking the spatial organization of the origins and their activity. Here, we show that Fkh1 and Fkh2 share a unique structural feature of human FoxP proteins that enables FoxP2 and FoxP3 to form domain-swapped dimers capable of bridging two DNA molecules in vitro. Accordingly, Fkh1 self-associates in vitro and in vivo in a manner dependent on the conserved domain-swapping region, strongly suggestive of homodimer formation. Fkh1- and Fkh2-domain-swap-minus (dsm) mutations are functional as transcription factors yet are defective in replication origin timing control. Fkh1-dsm binds replication origins in vivo but fails to cluster them, supporting the conclusion that Fkh1 and Fkh2 dimers perform a structural role in the spatial organization of chromosomal elements with functional importance.
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Proteínas de Ciclo Celular/metabolismo , Cromosomas Fúngicos/genética , Momento de Replicación del ADN , Factores de Transcripción Forkhead/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cromosomas Fúngicos/metabolismo , Dimerización , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/genética , Fase G1 , Regulación Fúngica de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Origen de Réplica , Fase S , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alineación de SecuenciaRESUMEN
Conformation capture technologies (e.g., Hi-C) chart physical interactions between chromatin regions on a genome-wide scale. However, the structural variability of the genome between cells poses a great challenge to interpreting ensemble-averaged Hi-C data, particularly for long-range and interchromosomal interactions. Here, we present a probabilistic approach for deconvoluting Hi-C data into a model population of distinct diploid 3D genome structures, which facilitates the detection of chromatin interactions likely to co-occur in individual cells. Our approach incorporates the stochastic nature of chromosome conformations and allows a detailed analysis of alternative chromatin structure states. For example, we predict and experimentally confirm the presence of large centromere clusters with distinct chromosome compositions varying between individual cells. The stability of these clusters varies greatly with their chromosome identities. We show that these chromosome-specific clusters can play a key role in the overall chromosome positioning in the nucleus and stabilizing specific chromatin interactions. By explicitly considering genome structural variability, our population-based method provides an important tool for revealing novel insights into the key factors shaping the spatial genome organization.
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Cromosomas/ultraestructura , Imagenología Tridimensional/métodos , Metagenómica/métodos , Animales , Evolución Biológica , Línea Celular , Centrómero/ultraestructura , Cromatina/genética , Cromatina/ultraestructura , Posicionamiento de Cromosoma , Cromosomas/genética , Cromosomas Humanos/genética , Cromosomas Humanos/ultraestructura , Diploidia , Genoma Humano , Heterocromatina/ultraestructura , Humanos , Hibridación Fluorescente in Situ , Funciones de Verosimilitud , Linfocitos/ultraestructura , Primates/genética , Análisis de la Célula Individual , Procesos Estocásticos , Tomografía por Rayos X/métodosRESUMEN
Gene duplication has a central role in evolution; still, little is known on the fates of the duplicated copies, their relative frequency, and on how environmental conditions affect them. Moreover, the lack of rigorous definitions concerning the fate of duplicated genes hinders the development of a global vision of this process. In this paper, we present a new framework aiming at characterizing and formally differentiating the fate of duplicated genes. Our framework has been tested via simulations, where the evolution of populations has been simulated using aevol, an in silico experimental evolution platform. Our results show several patterns that confirm some of the conclusions from previous studies, while also exhibiting new tendencies; this may open up new avenues to better understand the role of duplications as a driver of evolution.
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High resolution cellular signal encoding is critical for better understanding of complex biological phenomena. DNA-based biosignal encoders alter genomic or plasmid DNA in a signal dependent manner. Current approaches involve the signal of interest affecting a DNA edit by interacting with a signal specific promoter which then results in expression of the effector molecule (DNA altering enzyme). Here, we present the proof of concept of a biosignal encoding system where the enzyme terminal deoxynucleotidyl transferase (TdT) acts as the effector molecule upon directly interacting with the signal of interest. A template independent DNA polymerase (DNAp), TdT incorporates nucleotides at the 3' OH ends of DNA substrate in a signal dependent manner. By employing CRISPR-Cas9 to create double stranded breaks in genomic DNA, we make 3'OH ends available to act as substrate for TdT. We show that this system can successfully resolve and encode different concentrations of various biosignals into the genomic DNA of HEK-293T cells. Finally, we develop a simple encoding scheme associated with the tested biosignals and encode the message "HELLO WORLD" into the genomic DNA of HEK-293T cells at a population level with 91% accuracy. This work demonstrates a simple and engineerable system that can reliably store local biosignal information into the genomes of mammalian cell populations.
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Key to understanding many biological phenomena is knowing the temporal ordering of cellular events, which often require continuous direct observations [1, 2]. An alternative solution involves the utilization of irreversible genetic changes, such as naturally occurring mutations, to create indelible markers that enables retrospective temporal ordering [3-8]. Using NSC-seq, a newly designed and validated multi-purpose single-cell CRISPR platform, we developed a molecular clock approach to record the timing of cellular events and clonality in vivo , while incorporating assigned cell state and lineage information. Using this approach, we uncovered precise timing of tissue-specific cell expansion during murine embryonic development and identified new intestinal epithelial progenitor states by their unique genetic histories. NSC-seq analysis of murine adenomas and single-cell multi-omic profiling of human precancers as part of the Human Tumor Atlas Network (HTAN), including 116 scRNA-seq datasets and clonal analysis of 418 human polyps, demonstrated the occurrence of polyancestral initiation in 15-30% of colonic precancers, revealing their origins from multiple normal founders. Thus, our multimodal framework augments existing single-cell analyses and lays the foundation for in vivo multimodal recording, enabling the tracking of lineage and temporal events during development and tumorigenesis.
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Here we present an approach that combines a clustered regularly interspaced short palindromic repeats (CRISPR) system that simultaneously targets hundreds of epigenetically diverse endogenous genomic sites with high-throughput sequencing to measure Cas9 dynamics and cellular responses at scale. This massive multiplexing of CRISPR is enabled by means of multi-target guide RNAs (mgRNAs), degenerate guide RNAs that direct Cas9 to a pre-determined number of well-mapped sites. mgRNAs uncovered generalizable insights into Cas9 binding and cleavage, revealing rapid post-cleavage Cas9 departure and repair factor loading at protospacer adjacent motif-proximal genomic DNA. Moreover, by bypassing confounding effects from guide RNA sequence, mgRNAs unveiled that Cas9 binding is enhanced at chromatin-accessible regions, and cleavage by bound Cas9 is more efficient near transcribed regions. Combined with light-mediated activation and deactivation of Cas9 activity, mgRNAs further enabled high-throughput study of the cellular response to double-strand breaks with high temporal resolution, revealing the presence, extent (under 2 kb) and kinetics (~1 h) of reversible DNA damage-induced chromatin decompaction. Altogether, this work establishes mgRNAs as a generalizable platform for multiplexing CRISPR and advances our understanding of intracellular Cas9 activity and the DNA damage response at endogenous loci.
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Sistemas CRISPR-Cas , ARN Guía de Kinetoplastida , Cromatina/genética , ADN/metabolismo , Reparación del ADN/genética , Genómica , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismoRESUMEN
Robust SNP genotyping technologies and data analysis programs have encouraged researchers in recent years to use SNPs for linkage studies. Platforms used to date have been 10 K chip arrays, but the possible value of interrogating SNPs at higher densities has been considered. Here, we present a genome-wide linkage analysis by means of a 500 K SNP platform. The analysis was done on a large pedigree affected with Parkinsonian-pyramidal syndrome (PPS), and the results showed linkage to chromosome 22. Sequencing of candidate genes revealed a disease-associated homozygous variation (R378G) in FBXO7. FBXO7 codes for a member of the F-box family of proteins, all of which may have a role in the ubiquitin-proteosome protein-degradation pathway. This pathway has been implicated in various neurodegenerative diseases, and identification of FBXO7 as the causative gene of PPS is expected to shed new light on its role. The performance of the array was assessed and systematic analysis of effects of SNP density reduction was performed with the real experimental data. Our results suggest that linkage in our pedigree may have been missed had we used chips containing less than 100,000 SNPs across the genome.
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Enfermedad de Parkinson/genética , Polimorfismo de Nucleótido Simple , Tractos Piramidales , Sustitución de Aminoácidos , Cromosomas Humanos Par 22/genética , Proteínas F-Box/genética , Femenino , Ligamiento Genético , Genoma Humano , Humanos , Escala de Lod , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Linaje , Fenotipo , Mutación Puntual , Sinapsinas/genética , SíndromeRESUMEN
Classic approaches to mapping the developmental history of cells in vivo have relied on techniques that require complex interventions and often capture only a single trajectory or moment in time. We have previously described a developmental barcoding system to address these issues using synthetically induced mutations to record information about each cell's lineage in its genome. This system uses MARC1 mouse lines, which have multiple homing guide RNAs that each generate hundreds of mutant alleles and combine to produce an exponential diversity of barcodes. Here, we detail two MARC1 lines that are available from a public repository. We describe strategies for using MARC1 mice and experimental design considerations. We provide a protocol for barcode retrieval and sequencing as well as the analysis of the sequencing data. This protocol generates barcodes based on synthetically induced mutations in mice to enable lineage analysis.
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Sistemas CRISPR-Cas/genética , Código de Barras del ADN Taxonómico/métodos , Filogenia , Animales , Ratones , Mutación/genética , ARN Guía de Kinetoplastida/genéticaRESUMEN
The host factor, nuclear factor of activated T-cells (NFAT), regulates the transcription and replication of HIV-1. Here, we have determined the crystal structure of the DNA binding domain of NFAT bound to the HIV-1 long terminal repeat (LTR) tandem kappaB enhancer element at 3.05 A resolution. NFAT binds as a dimer to the upstream kappaB site (Core II), but as a monomer to the 3' end of the downstream kappaB site (Core I). The DNA shows a significant bend near the 5' end of Core I, where a lysine residue from NFAT bound to the 3' end of Core II inserts into the minor groove and seems to cause DNA bases to flip out. Consistent with this structural feature, the 5' end of Core I become hypersensitive to dimethylsulfate in the in vivo footprinting upon transcriptional activation of the HIV-1 LTR. Our studies provide a basis for further investigating the functional mechanisms of NFAT in HIV-1 transcription and replication.
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Elementos de Facilitación Genéticos , Duplicado del Terminal Largo de VIH/fisiología , VIH-1/genética , FN-kappa B/genética , Factores de Transcripción NFATC/metabolismo , Sitios de Unión , Simulación por Computador , Cristalografía por Rayos X , ADN/química , Humanos , Modelos Moleculares , FN-kappa B/metabolismo , Factores de Transcripción NFATC/química , Factores de Transcripción NFATC/genética , Conformación de Ácido Nucleico , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
DNA is an emerging medium for digital data and its adoption can be accelerated by synthesis processes specialized for storage applications. Here, we describe a de novo enzymatic synthesis strategy designed for data storage which harnesses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) in kinetically controlled conditions. Information is stored in transitions between non-identical nucleotides of DNA strands. To produce strands representing user-defined content, nucleotide substrates are added iteratively, yielding short homopolymeric extensions whose lengths are controlled by apyrase-mediated substrate degradation. With this scheme, we synthesize DNA strands carrying 144 bits, including addressing, and demonstrate retrieval with streaming nanopore sequencing. We further devise a digital codec to reduce requirements for synthesis accuracy and sequencing coverage, and experimentally show robust data retrieval from imperfectly synthesized strands. This work provides distributive enzymatic synthesis and information-theoretic approaches to advance digital information storage in DNA.
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Apirasa/metabolismo , ADN Nucleotidilexotransferasa/metabolismo , ADN/síntesis química , Almacenamiento y Recuperación de la Información/métodos , Nanoporos , Análisis de Secuencia de ADNRESUMEN
PURPOSE: The gene coding cytochrome P4501B1 (CYP1B1) has been shown to be a major cause of primary congenital glaucoma in the Iranian population. More recently it was shown to also be important in juvenile-onset open angle glaucoma (JOAG). We aimed to further investigate the role of CYP1B1 in a larger cohort of primary open angle glaucoma (POAG) patients which included late-onset patients. We also aimed to set up a microarray based protocol for mutation screening with an intent of using the protocol in a future population level screening program. METHODS: Sixty three POAG patients, nine affected family members, and thirty three previously genotyped primary congenital glaucoma (PCG) patients were included in the study. Clinical examination included slit lamp biomicroscopy, IOP measurement, gonioscopic evaluation, fundus examination, and measurement of perimetry. G61E, R368H, R390H, and R469W were screened by a protocol that included multiplexed allele specific amplification in the presence of a protease (PrASE), use of sequence tagged primers, and hybridization to generic arrays on microarray slides. The entire coding sequences of CYP1B1 and myocilin (MYOC) genes were sequenced in all individuals assessed by the microarray assay to carry a mutation. Intragenic single nucleotide polymorphism (SNP) haplotpes were determined for mutated alleles. RESULTS: Genotypes assessed by the array-based PrASE methodology were in 100% concordance with sequencing results. Seven mutation carrying POAG patients (11.1%) were identified, and their distribution was quite skewed between the juvenile-onset individuals (5/21) as compared to late-onset cases (2/42). Four of the seven mutation carrying Iranian patients harbored two mutated alleles. CYP1B1 mutated alleles in Iranian PCG and POAG patients shared common haplotypes. MYOC mutations were not observed in any of the patients. CONCLUSIONS: The PrASE approach allowed reliable simultaneous genotyping of many individuals. It can be an appropriate tool for screening common mutations in large sample sizes. The results suggest that CYP1B1 is implicated in POAG among Iranians, notably in the juvenile-onset form. Contrary to POAG patients studied in other populations, many mutation harboring Iranian patients carry two mutated alleles. We propose an explanation for this observation.
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Sistema Enzimático del Citocromo P-450/genética , Pruebas Genéticas , Glaucoma de Ángulo Abierto/enzimología , Glaucoma de Ángulo Abierto/genética , Mutación/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Péptido Hidrolasas/metabolismo , Adolescente , Adulto , Anciano , Hidrocarburo de Aril Hidroxilasas , Pueblo Asiatico/genética , Secuencia de Bases , Niño , Citocromo P-450 CYP1B1 , Análisis Mutacional de ADN , Femenino , Genotipo , Humanos , Irán , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Linaje , FenotipoRESUMEN
In vivo barcoding using nuclease-induced mutations is a powerful approach for recording biological information, including developmental lineages; however, its application in mammalian systems has been limited. We present in vivo barcoding in the mouse with multiple homing guide RNAs that each generate hundreds of mutant alleles and combine to produce an exponential diversity of barcodes. Activation upon conception and continued mutagenesis through gestation resulted in developmentally barcoded mice wherein information is recorded in lineage-specific mutations. We used these recordings for reliable post hoc reconstruction of the earliest lineages and investigation of axis development in the brain. Our results provide an enabling and versatile platform for in vivo barcoding and lineage tracing in a mammalian model system.
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Sistemas CRISPR-Cas , Desarrollo Embrionario/genética , Perfilación de la Expresión Génica/métodos , Alelos , Animales , Linaje de la Célula , Células Madre Embrionarias , Ratones , Mutación , ARN Guía de Kinetoplastida/genéticaRESUMEN
PURPOSE: To perform a mutation screening of TACSTD2 in 13 Iranian Gelatinous Drop-like Corneal Dystrophy (GDLD) pedigrees. To assess genotype-phenotype correlations. To determine intragenic SNP haplotypes associated with the mutations, so as to gain information on their origin. METHODS: The coding region of TACSTD2 was sequenced in the probands of 13 unrelated Iranian GDLD pedigrees. Variations were assessed in other available affected and unaffected family members and in unrelated normal control subjects by restriction fragment length polymorphism (RFLP). The variations were classified as being associated with disease if they segregated with the disease phenotype in the families, were not observed in 100 control individuals, disrupted protein expression, or affected conserved positions in the coded protein. Three intragenic single-nucleotide polymorphisms (SNPs) were used to define haplotypes associated with putative disease-causing mutations. RESULTS: The probands were each homozygous for one of four putative disease-causing variations observed in TACSTD2: C66X, F114C, L186P, and E227K. Three of these are novel. E227K was found in 10 of the Iranian patients. There were some phenotypic differences among different patients carrying this mutation-for example, with respect to age at onset. Genotyping of intragenic SNPs identified four haplotypes. C66X, F114C, and L186P were each associated with a haplotype common among control chromosomes, whereas all E227K alleles were associated with a haplotype not found among the control chromosomes. CONCLUSIONS: Although mutations in TACSTD2 among Iranian patients with GDLD were heterogeneous, E227K was found to be a common mutation. It is suggested that E227K may be a founder mutation in this population. Based on positions of known mutations in TACSTD2, significance of the thyroglobulin domain of the TACSTD2 protein in the pathogenesis of GDLD is suggested.
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Amiloidosis/genética , Antígenos de Neoplasias/genética , Moléculas de Adhesión Celular/genética , Distrofias Hereditarias de la Córnea/genética , Efecto Fundador , Mutación , Adolescente , Adulto , Edad de Inicio , Niño , Análisis Mutacional de ADN , Femenino , Genotipo , Haplotipos , Humanos , Irán , Masculino , Persona de Mediana Edad , Linaje , Fenotipo , Polimorfismo de Longitud del Fragmento de Restricción , Polimorfismo de Nucleótido SimpleRESUMEN
Cutis laxa is a rare group of inherited and acquired disorders characterized by loose and redundant skin with reduced elasticity. Mutations in the elastin coding gene have been shown to cause autosomal dominant cutis laxa in three families. A homozygous mutation in the fibulin-5 coding gene was discovered in a Turkish pedigree showing recessive inheritance, and a different mutation in this gene was found in the heterozygous state in a sporadic case of the disease. Here, we report the third case of a mutation in the fibulin-5 coding gene in a recessive Iranian cutis laxa pedigree. The mutation is the same as previously reported in the Turkish pedigree, further confirming that it is causative of disease. A haplotype consisting of seven intragenic sequence variations common to both pedigrees is described for the mutation-carrying fibulin-5 allele.
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Cutis Laxo/genética , Proteínas de la Matriz Extracelular/genética , Genes Recesivos/genética , Mutación Missense/genética , Adolescente , Cutis Laxo/diagnóstico , Análisis Mutacional de ADN , Proteínas de la Matriz Extracelular/fisiología , Femenino , Ligamiento Genético , Haplotipos/genética , Homocigoto , Humanos , Irán , Masculino , Linaje , TurquíaRESUMEN
RNA-sequencing (RNA-seq) measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. In contrast, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq, our method enriches for context-specific transcripts over housekeeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d.
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Perfilación de la Expresión Génica/métodos , ARN Mensajero/genética , Análisis de Secuencia de ARN/métodos , ADN Complementario/genética , Fluorescencia , Microscopía Confocal/métodosRESUMEN
Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked complementary DNA (cDNA) amplicons are sequenced within a biological sample. Using 30-base reads from 8102 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay. FISSEQ is compatible with tissue sections and whole-mount embryos and reduces the limitations of optical resolution and noisy signals on single-molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ.