lysis and digestion of scarce cells

Un enfoque integral para estudiar el interactoma promotor en células escasas

Temporal gene expression drives cell differentiation and, ultimately, organism development, and its alteration is closely related to a wide plethora of diseases1,2,3,4,5. Gene transcription is finely regulated by the action of regulatory elements, which can be classified as proximal (i.e., gene promoters) and distal (e.g., enhancers or silencers), the latter of which are frequently located afar from their target genes and physically interact with them through chromatin looping to modulate gene expression6,7,8.
The identification of distal regulatory regions in the genome is a matter which is widely agreed upon, since these regions harbor specific histone modifications9,10,11 and contain specific transcription factor recognition motifs, acting as recruiting platforms for them12,13,14. Besides, in the case of enhancers and super-enhancers15,16, they also have low-nucleosome occupancy17,18 and are transcribed into non-coding eRNAs19,20.
Nonetheless, each distal regulatory element&#39;s target genes are more difficult to predict. More often than not, interactions between distal regulatory elements and their targets are cell-type and stimulus specific21,22, span hundreds of kilobases, bridging over other genes in any direction23,24,25, and can even be located inside intronic regions of their target gene or other non-intervening genes26,27. Furthermore, distal regulatory elements can also control more than one gene at the same time, and vice versa28,29. This positional complexity hinders pinpointing regulatory associations between them, and therefore, most of each regulatory element&#39;s targets in every cell type remain unknown.
During recent years, there has been a significant boom in the development of chromosome conformation capture (3C) techniques for studying chromatin interactions. The most widely used of them, Hi-C, allows to generate a map of all the interactions between every fragment of a cell&#39;s genome30. However, to detect significant interactions at the restriction fragment level, Hi-C relies on ultra-deep sequencing, prohibiting its use to routinely study the regulatory landscape of individual genes. To overcome this economic limitation, several enrichment-based 3C techniques have emerged, such as ChIA-PET31, HiChIP32, and its low-input counterpart HiCuT33. These techniques depend on the use of antibodies to enrich for genome-wide interactions mediated by a specific protein. Nonetheless, the unique feature of these 3C techniques is also the bane of their application; users count on the availability of high-quality antibodies for the protein of interest and cannot compare conditions in which the binding of the protein is dynamic.
Promoter Capture Hi-C (PCHi-C) is another enrichment-based 3C technique that circumvents these limitations34,35. By employing a biotinylated RNA probe enrichment system, PCHi-C is able to generate genome-wide high-resolution libraries of genomic regions interacting with 28,650 human- or 27,595 mouse-annotated gene promoters, also known as the promoter interactome. This approach allows one to detect significant long-range interactions at the restriction fragment level resolution of both active and inactive promoters, and robustly compare promoter interactomes between any condition independently of the dynamics of histone modifications or protein binding. PCHi-C has been widely used over recent years to identify promoter interactome reorganizations during cell differentiation36,37, identify the mechanism of action of transcription factors38,39, and discover new potential genes and pathways deregulated in disease by non-coding variants40,41,42,43,44,45,46,47,48, alongside new driver non-coding mutations49,50. Besides, by just modifying the capture system, this technique can be customized according to the biological question to interrogate any interactome (e.g., the enhancer interactome51 or the interactome of a collection of non-coding alterations41,52).
However, PCHi-C relies on a minimum of 20 million cells to perform the technique, which prevents the study of scarce cell populations such as the ones often used in developmental biology and clinical applications. For this reason, we have developed low input Capture Hi-C (liCHi-C), a new cost-effective and customizable method based on the experimental framework of PCHi-C to generate high-resolution promoter interactomes with low-cell input. By performing the experiment with minimal tube changes, swapping or eliminating steps from the original PCHi-C protocol, drastically reducing reaction volumes, and modifying reagent concentrations, library complexity is maximized and it is possible to generate high-quality libraries with as little as 50,000 cells53.
Low input Capture Hi-C (liCHi-C) has been benchmarked against PCHi-C and used to elucidate promoter interactome rewiring during human hematopoietic cell differentiation, discover potential new disease-associated genes and pathways deregulated by non-coding alterations, and detect chromosomal abnormalities53. The step-by-step protocol and the different quality controls through the technique are detailed here until the final generation of the libraries and their computational analysis.

Autor destacado: Un flujo de trabajo integrado para estudiar la arquitectura del genoma espacio-temporal centrada en el promotor en poblaciones celulares escasas  

Un flujo de trabajo integrado para estudiar la arquitectura del genoma espacio-temporal centrada en el promotor en poblaciones celulares escasas

Watch this Scientific Journal Video about Lysis and Digestion of Scarce Cells at JoVE.com

Lysis and Digestion of Scarce Cells  

Lisis y digestión de células escasas

Para comenzar, tome una alícuota de células escasas congeladas y agregue 1 mililitro de tampón de lisis fría para interrumpir la membrana celular. Incubar las células en hielo durante 30 minutos, mezclando por inversión cada cinco minutos. Centrifugar los núcleos durante 10 minutos a 1.000 G.Retire el sobrenadante y vuelva a suspender los núcleos en 500 microlitros de tampón de restricción frío 1.25X 2.Luego agregue 5.5 microlitros de dodecil sulfato de sodio al 10%, mezcle por vórtice e incube en un bloque térmico. Luego apague la SDS agregando 37.5 microlitros de 10% Triton X-100. Mezclar e incubar la muestra como se demostró anteriormente.Añadir 7,5 microlitros de Hind III para digerir la cromatina mezclar la solución e incubar durante la noche a 37 grados centígrados.

Research

JoVE Journal

Genetics

Lysis and Digestion of Scarce Cells

218 vistas. Para comenzar, tome una alícuota de células escasas congeladas y agregue 1 mililitro de tampón de lisis fría para interrumpir la membrana celular. Incubar las células en hielo durante 30 minutos, mezclando por inversión cada cinco minutos. Centrifugar los núcleos durante 10 minutos a 1.000 G.Retire el sobrenadante y vuelva a suspender los núcleos en 500 microlitros de tampón de restricción frío 1.25X 2.Luego agregue 5.5 microlitros de dodecil sulfato de sodio al 10%, mezcle ...

Video: Lysis and Digestion of Scarce Cells  

An Integrated Workflow to Study the Promoter-Centric Spatio-Temporal Genome Architecture in Scarce Cell Populations

Spatiotemporal gene transcription is tightly regulated by distal regulatory elements, such as enhancers and silencers, which rely on physical proximity with their target gene promoters to control transcription. Although these regulatory elements are easy to identify, their target genes are difficult to predict, since most of them are cell-type specific and may be separated by hundreds of kilobases in the linear genome sequence, skipping over other non-target genes. For several years, Promoter Capture Hi-C (PCHi-C) has been the gold standard for the association of distal regulatory elements to their target genes. However, PCHi-C relies on the availability of millions of cells, prohibiting the study of rare cell populations such as those commonly obtained from primary tissues. To overcome this limitation, low input Capture Hi-C (liCHi-C), a cost-effective and customizable method to identify the repertoire of distal regulatory elements controlling each gene of the genome, has been developed. liCHi-C relies on a similar experimental and computational framework as PCHi-C, but by employing minimal tube changes, modifying&#160;the reagent concentration and volumes, and swapping or eliminating steps, it accounts&#160;for minimal material loss during library construction. Collectively, liCHi-C enables the study of gene regulation and spatiotemporal genome organization in the context of developmental biology and cellular function.

Gene expression is regulated by interactions of gene promoters with distal regulatory elements. Here, we descirbe how low input Capture Hi-C (liCHi-C) allows the identification of these interactions in rare cell types, which were previously unmeasurable.

Spatiotemporal gene transcription is tightly regulated by distal regulatory elements, such as enhancers and silencers, which rely on physical proximity ...

Investigación

Genética

Ligation and Decrosslinking of Scarce Cell DNA

Ligadura y desentrecruzamiento de ADN de células escasas

Sonication and Biotin Pull-Down of Scarce Cells DNA

Sonicación y extracción de biotina de ADN de células escasas

dATP-Tailing, Adapter Ligation, and PCR Amplification of Scarce Cell DNA

DATP-Tailing, Adapter Ligation, and PCR Amplification of Scarce Cell DNA  

dATP-Tailing, ligadura de adaptador y amplificación por PCR de ADN de células escasas

Library Capture, Biotin Pull-Down, and PCR Amplification Using Scarce Cell DNA Library

Captura de biblioteca, extracción de biotina y amplificación por PCR utilizando la biblioteca de ADN de células escasas