lysis and digestion of scarce cells

A Comprehensive Approach to Studying the Promoter Interactome in Scarce Cells

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.

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

An Integrated Workflow to Study the Promoter-Centric 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 Lysis and Digestion of Scarce Cells at JoVE.com

Lysis and Digestion of Scarce Cells  

To begin, take an aliquot of frozen scarce cells and add 1 milliliter of cold lysis buffer to disrupt the cell membrane. Incubate the cells on ice for 30 minutes, mixing by inversion every five minutes. Centrifuge the nuclei for 10 minutes at 1, 000 G.Remove the supernatant and re-suspend the nuclei in 500 microliters of cold 1.25X restriction buffer 2.Then add 5.5 microliters of 10%sodium dodecyl sulfate, mix by vortexing and incubate in a thermo block. Then quench the SDS by adding 37.5 microliters of 10%Triton X-100. Mix and incubate the sample as demonstrated earlier.Add 7.5 microliters of Hind III to digest the chromatin mix the solution and incubate overnight at 37 degrees Celsius.

Research

JoVE Journal

Genetics

218 Views.  Josep Carreras Leukaemia Research Institute. Per iniziare, prendi un'aliquota di cellule scarse congelate e aggiungi 1 millilitro di tampone di lisi fredda per interrompere la membrana cellulare. Incubare le cellule sul ghiaccio per 30 minuti, mescolando per inversione ogni cinque minuti. Centrifugare i nuclei per 10 minuti a 1.000 G.Rimuovere il surnatante e risospendere i nuclei in 500 microlitri di tampone di restrizione freddo 1,25X 2.Quindi aggiungere 5,5 microlitri di sodio dodecilsolfato al 10%, mescolare a vortice e incub...

Video: Lysis and Digestion of Scarce Cells  

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 ...

Ligation and Decrosslinking of Scarce Cell DNA

To begin, take the digested scarce cell's DNA and place it on ice. Prepare a master mix by using the reagents required to fill in and biotinylate the restriction fragment overhangs. Incubate the sample at 37 degrees Celsius for 75 minutes, mixing by inversion every 15 minutes.Chill the sample on ice, and ligate the filled-in DNA ends by preparing a master mix using the reagents required for ligation. Finally, incubate the sample for four to six hours at 16 degrees Celsius, and then for 30 minutes at room temperature. Add 30 microliters of 10 milligrams per milliliter of Proteinase K to decrosslink the chromatin.Mix the solution, and incubate overnight at 65 degrees Celsius.

Sonication and Biotin Pull-Down of Scarce Cells DNA

After purifying the DNA, begin by taking ligated and decross-linked digested chromatin of the scarce cells. Set the water bath sonicator at a duty factor of 20%peak incidence power of 50 to 200 cycles per burst for 65 seconds, and a temperature range of 6 to 10 degrees Celsius, and sonicate the ligated and decross-linked digested chromatin. After end repairing the sample, transfer 150 microliters of C1 streptavidin beads per sample to a 1.5 milliliter tube.Place them on a 1.5 milliliter tube magnet and wait until all the beads are stuck to the wall. Then remove the supernatant, leaving the beads behind. Next, wash the beads by adding 400 microliters of Tween buffer and resuspending them with soft vortexing.Place the tube back in the magnet until all the beads are stuck to the wall. Then remove the supernatant, leaving the beads behind. After resuspending the beads, in 300 microliters of 2X No-Tween buffer or NTB, combine them with the 300 microliters of the sample.Finally, incubate for 15 minutes rotating at room temperature to pull down the informative DNA fragments with biotin.

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

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

To begin, take the scarce cell's DNA fragments pulled with biotin. Prepare a master mix by adding the reagents for dATP tailing and incubate the sample for 30 minutes at 37 degrees Celsius. Then, inactivate Klenow exo-minus by incubating the sample for 10 minutes at 65 degrees Celsius and cooling it on ice.After washing the beads with 300 microliters each of TB, NTB, and 100 microliters of ligation buffer, resuspend them in 50 microliters of ligation buffer. Add four microliters of 15 micromolar pre-annealed adapter mix and one microliter of 2000 units per microliter T4 DNA ligase to the sample. Wash the beads with 400 microliters of TB, 200 microliters of NTB, and 100 microliters of restriction buffer two.After amplifying the library by PCR, pool all the 50 microliter reactions from the same sample into a 1.5 milliliter DNA low binding tube. Place it on a tube magnet and wait until all the beads are stuck to the wall. Transfer the supernatant containing the library to a new 1.5 milliliter DNA low binding tube and top up with TLE buffer to 500 microliters.To perform a double-sided selection using paramagnetic bead purification, add 200 microliters of stock beads to the library, mix by vortexing, and incubate for 10 minutes, rotating at room temperature. Then, place the library on a magnet and wait until all the beads are stuck to the wall. Transfer the supernatant containing the library to a new 1.5 milliliter DNA low binding tube.Concentrate the beads by taking 750 microliters of stock beads and placing them in a 1.5 milliliter tube on a magnet. Once all the beads are stuck to the wall, remove the supernatant and resuspend the beads by vortexing in 300 microliters of new stock beads. Add 300 microliters of concentrated beads to the sample.Mix by vortexing and incubate for 10 minutes, rotating at room temperature. Place on a magnet. Wait until all the beads are stuck to the wall and remove the supernatant.Wash the beads by adding one milliliter of 70%ethanol to the tube with the beads still on the magnet. Allow the beads to air dry and resuspend them in 21 microliters of TLE buffer by vortexing. To elute the library from the beads, incubate the sample for 10 minutes at 37 degrees Celsius in a thermoblock.Place the tube in a magnet and transfer the supernatant containing the library to a new 1.5 milliliter DNA low binding tube.

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

Begin by taking 500 to 1000 nanograms of the scarce cell DNA library. Dry the DNA with a vacuum concentrator and resuspended it in 3.4 microliters of nuclease-free water. After adding the blockers, and while on the thermocycler, at 65 degrees Celsius, transfer the hybridization solution with the biotinylated RNA to the blocked library.After closing the tube lid firmly, incubate in the thermocycler overnight at 65 degrees Celsius. Then transfer 50 microliters of T1 Streptavidin beads per sample to a 1.5 milliliter DNA low binding tube and place them on a 1.5 milliliter tube magnet. Once all the beads are stuck to the wall, remove the supernatant leaving the beads behind.Now wash the beads thrice with 200 microliters of the binding buffer from the target enrichment kit and resuspend the beads in 200 microliters of binding buffer. Transfer the sample to the resuspended beads while on the thermocycler and incubate for 30 minutes. After washing the beads with 200 microliters of wash buffer 1, incubate them for 15 minutes, rotating at room temperature.Then wash the beads thrice with 200 microliters of wash buffer 2 heated to 65 degrees Celsius and incubate in a thermo block. Finally, after amplifying the library by PCR, perform a DNA purification using paramagnetic beads by adding 270 microliters of stock beads to the sample and mixing by vortexing. An automated electrophoresis profile from a library, just before capture, provided 49.7 nanograms per microliter of DNA in 20 microliters reaction volume.However, 3.06 nanograms per microliter of DNA is obtained from a high sensitivity automated electrophoresis profile from a finished leachic library.