Name: Ligation and Decrosslinking of Scarce Cell DNA
Uploaded: 2023-04-21T14:20:00
Description: Watch this Scientific Journal Video about Ligation and Decrosslinking of Scarce Cell DNA at JoVE.com

ligation and decrosslinking of scarce cell dna

희소한 세포에서 프로모터 상호작용체를 연구하기 위한 포괄적인 접근법

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.

저자 스포트라이트: 희소한 세포 집단에서 프로모터 중심의 시공간 게놈 아키텍처를 연구하기 위한 통합 워크플로우  

희소한 세포 집단에서 프로모터 중심의 시공간 게놈 구조를 연구하기 위한 통합 워크플로우An Integrated Workflow to Study the promoter-centic spatio-temporal genome architecture in Scarce Cell Populations

With liCHi-C, the main question that we are trying to answer is how the chromatin interacts in the nucleus, and therefore, to understand a new layer of gene regulation. We have proven with liCHi-C that we can explore the genome organization of a given sample, link the non-coding mutation associated with disease with potential genes affected, and detect chromosomal rearrangements, such as translocation, for example, in tumor samples. Now we can explore the 3D chromatin organization of scar cell types, such as primary stem cells or relevant clinical samples.Our protocol reduces the input materials and the time and cost needed to obtain quality data to explore the genome organization at the restriction fragment resolution. liCHi-C is expanding the reach of the 3D chromatin organization field, allowing us to explore new cell types previously impossible to analyze. Thanks to liCHi-C, the scientific community can explore the special regulation of gene expression.For example, in malignant transformation only in a specific clonal sub-population inside a tumor.

Watch this Scientific Journal Video about Ligation and Decrosslinking of Scarce Cell DNA at JoVE.com

Ligation and Decrosslinking of Scarce Cell DNA  

희소 세포 DNA의 결찰 및 탈가교

시작하려면 소화된 희소 세포의 DNA를 얼음 위에 올려 놓으십시오. 제한 단편 돌출부를 채우고 비오티닐화하는 데 필요한 시약을 사용하여 마스터 믹스를 준비합니다. 샘플을 섭씨 37도에서 75분 동안 배양하고 15분마다 반전하여 혼합합니다.샘플을 얼음 위에서 식히고 결찰에 필요한 시약을 사용하여 마스터 믹스를 준비하여 채워진 DNA 말단을 결찰합니다. 마지막으로 샘플을 섭씨 16도에서 4-6시간 동안 배양한 다음 실온에서 30분 동안 배양합니다. 염색질을 탈가교하기 위해 Proteinase K 밀리리터당 10밀리그램의 30마이크로리터를 추가합니다.용액을 혼합하고 섭씨 65도에서 밤새 배양합니다.

Research

JoVE Journal

Genetics

Ligation and Decrosslinking of Scarce Cell DNA

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 ...

연구

유전학

Lysis and Digestion of Scarce Cells

희소 세포의 용해 및 소화

Sonication and Biotin Pull-Down of Scarce Cells DNA

희소 세포 DNA의 초음파 처리 및 비오틴 풀다운

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

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

희소 세포 DNA의 dATP-tailing, 어댑터 결찰 및 PCR 증폭

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