Name: Library Capture, Biotin Pull-Down, and PCR Amplification Using Scarce Cell DNA Library
Uploaded: 2023-04-21T14:20:00
Description: Watch this Scientific Journal Video about Library Capture, Biotin Pull-Down, and PCR Amplification Using Scarce Cell DNA Library at JoVE.com

library capture, biotin pull-down, and pcr amplification using scarce cell dna library

दुर्लभ कोशिकाओं में प्रमोटर इंटरएक्टोम का अध्ययन करने के लिए एक व्यापक दृष्टिकोण

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.

लेखक स्पॉटलाइट: दुर्लभ सेल आबादी में प्रमोटर-केंद्रित स्थानिक-लौकिक जीनोम वास्तुकला का अध्ययन करने के लिए एक एकीकृत वर्कफ़्लो  

दुर्लभ सेल आबादी में प्रमोटर-केंद्रित स्थानिक-अस्थायी जीनोम आर्किटेक्चर का अध्ययन करने के लिए एक एकीकृत वर्कफ़्लो

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 Library Capture, Biotin Pull-Down, and PCR Amplification Using Scarce Cell DNA Library at JoVE.com

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

दुर्लभ सेल डीएनए लाइब्रेरी का उपयोग करके लाइब्रेरी कैप्चर, बायोटिन पुल-डाउन और पीसीआर प्रवर्धन

दुर्लभ सेल डीएनए लाइब्रेरी के 500 से 1000 नैनोग्राम लेने से शुरू करें। डीएनए को वैक्यूम कंसंट्रेटर से सुखाएं और इसे 3.4 माइक्रोलीटर न्यूक्लियस-मुक्त पानी में फिर से निलंबित करें। ब्लॉकर्स जोड़ने के बाद, और थर्मोसाइकलर पर रहते हुए, 65 डिग्री सेल्सियस पर, बायोटिनीलेटेड आरएनए के साथ संकरण समाधान को अवरुद्ध पुस्तकालय में स्थानांतरित करें।ट्यूब ढक्कन को मजबूती से बंद करने के बाद, थर्मोसाइकलर में रात भर 65 डिग्री सेल्सियस पर इनक्यूबेट करें। फिर प्रति नमूने टी 1 स्ट्रेप्टाविडिन मोतियों के 50 माइक्रोलीटर को 1.5 मिलीलीटर डीएनए कम बाध्यकारी ट्यूब में स्थानांतरित करें और उन्हें 1.5 मिलीलीटर ट्यूब चुंबक पर रखें। एक बार जब सभी मोती दीवार से चिपक जाते हैं, तो मोतियों को पीछे छोड़कर सतह पर तैरनेवाला को हटा दें।अब मोतियों को लक्ष्य संवर्धन किट से बाइंडिंग बफर के 200 माइक्रोलीटर के साथ तीन बार धोएं और 200 माइक्रोलीटर बाइंडिंग बफर में मोतियों को फिर से निलंबित करें। थर्मोसाइकलर पर रहते हुए नमूने को पुन: निलंबित मोतियों में स्थानांतरित करें और 30 मिनट के लिए इनक्यूबेट करें। 200 माइक्रोलीटर वॉश बफर 1 के साथ मोतियों को धोने के बाद, उन्हें कमरे के तापमान पर घूमते हुए 15 मिनट के लिए इनक्यूबेट करें।फिर मोतियों को 200 माइक्रोलीटर वॉश बफर 2 के साथ 65 डिग्री सेल्सियस तक गर्म करके तीन बार धोएं और थर्मो ब्लॉक में इनक्यूबेट करें। अंत में, पीसीआर द्वारा लाइब्रेरी को बढ़ाने के बाद, नमूने में 270 माइक्रोलीटर स्टॉक मोती जोड़कर और भंवर द्वारा मिश्रण करके पैरामैग्नेटिक बीड्स का उपयोग करके डीएनए शुद्धिकरण करें। कैप्चर से ठीक पहले एक पुस्तकालय से एक स्वचालित वैद्युतकणसंचलन प्रोफ़ाइल, 20 माइक्रोलीटर प्रतिक्रिया मात्रा में 49.7 नैनोग्राम प्रति माइक्रोलीटर डीएनए प्रदान करती है।हालांकि, 3.06 नैनोग्राम प्रति माइक्रोलीटर डीएनए एक तैयार लीच लाइब्रेरी से उच्च संवेदनशीलता स्वचालित वैद्युतकणसंचलन प्रोफ़ाइल से प्राप्त किया जाता है।

Research

JoVE Journal

Genetics

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

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 ...

Lysis and Digestion of Scarce Cells

दुर्लभ कोशिकाओं का लाइसिस और पाचन

Ligation and Decrosslinking of Scarce Cell DNA

दुर्लभ सेल डीएनए का बंधाव और डीक्रॉसलिंकिंग

Sonication and Biotin Pull-Down of Scarce Cells DNA

दुर्लभ कोशिकाओं डीएनए के सोनिकेशन और बायोटिन पुल-डाउन

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

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

डीएटीपी-टेलिंग, एडेप्टर लिगेशन, और दुर्लभ सेल डीएनए का पीसीआर प्रवर्धन