Name: single-cell suspension preparation from nile tilapia intestine for single-cell sequencing
Uploaded: 2023-02-10T14:00:00
Description: Watch this Scientific Journal Video about Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing at JoVE.com

The authors wish to acknowledge support from the Hainan Provincial Natural Science Foundation of China (NO. 320QN211) and the Research Fund Program of Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture of China (NO. PBEA2021ZD01).

All animal protocols during this study were approved by the Hainan University Institutional Animal Care and Use Committee (Protocol number: HNUAUCC-2022-00063; Approval date: 2022-03-03). A list of the equipment and supplies used in this experiment can be found in the Table of Materials. A summary of the current protocol is illustrated in Figure 1.
1. Fish preparation
<ol>
	<li>Obtain 6 month old Nile tilapia with a mean body we...

This protocol describes the preparation of a high-quality single-cell suspension of Nile tilapia intestine. Before dissociation, the removal of fat and mesentery from the intestine is necessary, particularly for carnivorous fish intestines with much fat. Using a syringe instead of hard scraping to wash off the intestinal contents reduces the mechanical damage to the cells. To ensure cell viability, it is also essential to maintain the temperature at 20 &#176;C or below for the tissue dissection and rinsing steps. The was...

Cells are the fundamental units of organisms. Compared to bulk-tissue studies, single-cell level studies can reflect cell heterogeneity and provide higher-resolution information1. In recent years, researchers have applied single-cell sequencing technologies for genome, transcriptome, epigenome, or multi-omic studies at the single-cell level in mammals, zebrafish, and other model organisms and reported great breakthroughs2,3,4,5,6,7. While most studies have focused on model organisms, there are few reference protocols or commercial dissociation kits for single-cell sequencing in economic fish species, which limits the application of single-cell sequencing in aquaculture research. Therefore, developing tissue dissociation protocols that produce high-quality single-cell suspensions with high cell viability and nucleic acid integrity is crucial.
Optimizing the tissue dissociation protocol with the appropriate enzyme or enzyme combination to obtain enough viable cells with minimum damage is essential. The most effective enzyme for tissue dissociation varies depending on the tissue type. In mammals, several enzymes have been used to prepare single-cell suspensions for mammalian solid tissues, including collagenase, dispase, trypsin, papain, elastase, hyaluronidase, liberase, accutase, and trypLE8,9. Trypsin digestion combined with mechanical disruption has commonly been used to dissociate tissues for cell culture in fish10,11,12,13,14. Trypsin has also been used or added into the digestion cocktail for tissue dissociation in the rat intestine15 and zebrafish gill tissue16. However, for several reasons, trypsin is not the best option for single-cell sequencing. Trypsin alone is usually ineffective for tissue dissociation. Additionally, trypsin induces DNA strand breaks17,18 and RNA degradation19.
Papain degrades the proteins that make up the tight junctions between cells. In mammalian nerve and smooth muscle cells, papain is more efficient and less destructive than other proteases20,21. However, like trypsin, papain results in the free DNA-induced aggregation of cells due to the cell lysis that occurs during enzymatic digestion9. Elastase breaks down elastin, which is typically found in the skin, lungs, ligaments, tendons, and vascular tissues22. It is often used in combination with collagenase, dispase, or trypsin for dissociating lung tissue8. Hyaluronidase cleaves the glycosidic bonds of hyaluronan, contributing to the digestion of the extracellular matrix in various connective tissues and skin9,23.
In general, collagenase and dispase are good options for extracellular matrix breakdown. They have been used in the dissociation of human, mouse, and zebrafish intestines24,25,26,27. Collagenase destroys the peptide bond in collagen, promotes the digestion of the extracellular matrix, and releases the cells into suspension, and, thus, collagenase is often used for human and mouse solid tissue dissociation, including for the liver28,29, spleen30, pancreas31, and intestine25. Dispase is a protease that hydrolyzes the N-terminal peptide bonds of nonpolar amino acid residues and is milder than collagenase. It cleaves the extracellular matrix components, such as fibronectin, type IV collagen, and to a lesser extent, type I collagen, without affecting cell-cell junctions. Dispase is used separately or with other enzymes for tissue dissociation, such as for intestine25,32, brain33, liver34, etc. In addition, commercially available digestion cocktails, including liberase, accutase, and trypLE, are also good alternatives for solid tissue dissociation, especially for the skin, liver, and kidney8,9.
Nile tilapia (Oreochromis niloticus) belongs to the family Cichlidae of the order Perciformes. It is one of the most cultured freshwater fish species in tropical and subtropical areas, with an annual production of 4.5 million tons in 202235. It is one of the best-studied aquaculture fish species with a well-annotated genome. Nile tilapia is an ideal research model for aquaculture fish species due to its short generation time, ease of rearing, and adaptability to a wide range of rearing environments. The intestine is of great research interest as it is the organ of nutrition digestion and absorption, metabolism, and mucosal immunity. The intestine is the habitat of microbial populations and is an essential immune tissue36. It is immunologically active due to the presence of numerous immune cell types, including macrophages, B cells, granulocytes, and T cells.
In the current study, we developed a protocol for preparing a high-quality single-cell suspension from the Nile tilapia intestine to facilitate single-cell level studies in aquaculture fish species. According to the characteristics of these tissue-specific enzymes and preliminary work, collagenase/dispase is appropriate for dissociating tilapia intestine tissue. The final enzyme type to consider in preparing single-cell suspensions is DNase-I, which prevents cell aggregation by degrading free DNA released through dead cell lysis during enzymatic digestion without initiating apoptotic pathways9 and increases the live cell yield36. Additionally, bovine serum albumin (BSA) is added to the washing buffer to reduce cell clumping and improve cell viability. Several reagent companies describe BSA as an enzyme stabilizer. The addition of 0.04%-1% BSA to PBS (phosphate-buffered saline) has been used to develop a washing solution for preparing single-cell sequencing suspensions without adverse effects38. The addition of a low ratio of BSA could help maintain cell viability and avoid the free DNA-induced aggregation of cells due to cell lysis. This protocol can also provide a valuable reference for developing cell dissociation protocols from the intestines of other aquaculture fish species.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22-&#956;m Sterile Filter</td>
			<td>Solarbio Life Sciences</td>
			<td>SLGV033RB</td>
			<td>It is used to filter and sterilize the enzyme solution.</td>
		</tr>
		<tr>
			<td>40-&#956;m Cell Strainer</td>
			<td>Solarbio Life Sciences</td>
			<td>F8200</td>
			<td>Cell Strainer is applied to eliminate undigested tissue pieces.</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>SRE0098</td>
			<td>Powder; dilute 0.04 g BSA with 100 mL 1&#215; DPBS to prepare 0.04% BSA-DPBS washing bffer. Store at 2 - 8 &#176;C.</td>
		</tr>
		<tr>
			<td>Collagenase II</td>
			<td>Sangon Biotech</td>
			<td>A004202</td>
			<td>Dilute with PBS to a final concentration of 1 mg/mL.</td>
		</tr>
		<tr>
			<td>Collagenase/dispase</td>
			<td>Roche</td>
			<td>10269638-001</td>
			<td>Dilute with PBS to a final concentration of 1 mg/mL.</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Sigma-Aldrich</td>
			<td>D4818</td>
			<td>Dilute with PBS to a final concentration of 1 mg/mL.</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma-Aldrich</td>
			<td>AMPD1</td>
			<td>DNase I helps reduce cell clumping.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline (DPBS), Ca2+/Mg2+-free</td>
			<td>Solarbio Life Sciences</td>
			<td>E607009-0500</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr>
			<td>Elastase</td>
			<td>Sangon Biotech</td>
			<td>A600438</td>
			<td>Dilute with PBS to a final concentration of 0.5 mg/mL.</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td>Serum, used at volume of 5% in digetstion solution.</td>
		</tr>
		<tr>
			<td>Inverted&#160;Microscope</td>
			<td>Leica</td>
			<td>qTOWER3G</td>
			<td>It is used to examine cell viability.</td>
		</tr>
		<tr>
			<td>Liberase</td>
			<td>Roche</td>
			<td>5401119001</td>
			<td>Dilute with PBS to a final concentration of 0.25 mg/mL.</td>
		</tr>
		<tr>
			<td>Nile tilpia (Oreochromis niloticus)</td>
			<td>ProGift Aquaculture Technology Co. Ltd.</td>
			<td>NA</td>
			<td>Healthy fish with no disease signs (Mean body weight: 100 g).&#160;</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Solarbio Life Sciences</td>
			<td>P1020</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr>
			<td>Refrigerated Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td>It is used to spin down the tissue and cell petet.</td>
		</tr>
		<tr>
			<td>RNase inhibitor</td>
			<td>NEB</td>
			<td>M0314L</td>
			<td>Inhibit RNase activity</td>
		</tr>
		<tr>
			<td>Solid-phase RNase-Be-Gone Reagent</td>
			<td>Sangon Biotech</td>
			<td>B644201-0050</td>
			<td>It is used to remove the RNase from tools such as dissecting scissors and glass pipettes. Store at room temperature.</td>
		</tr>
		<tr>
			<td>Tricaine methanesulfonate (MS-222)</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td>For fish euthanasia.&#160;</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Invitrogen</td>
			<td>C0040</td>
			<td>It is used for staining dead cells.</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sangon Biotech</td>
			<td>E607001</td>
			<td>Dilute with PBS to a final concentration of 1 mg/mL.</td>
		</tr>
	</tbody>
</table>

single-cell suspension preparation from nile tilapia intestine for single-cell sequencing

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The preparation of high-quality single-cell suspensions is essential for single-cell level studies such as single-cell RNA or genome sequencing. However, there is no ready-to-use protocol for aquaculture fish species, particularly for the intestine of tilapia. The effective dissociation enzymes vary depending on the tissue type. Therefore, optimizing the tissue dissociation protocol by selecting the appropriate enzyme or enzyme combination to obtain enough viable cells with minimum damage is essential. This study illustrates an optimized protocol to prepare a high-quality single-cell suspension from Nile tilapia intestine with an enzyme combination of collagenase/dispase. This combination is highly effective for dissociation with the utilization of bovine serum albumin and DNase to reduce cell aggregation after digestion. The cell output satisfies the requirements for single-cell sequencing, with 90% cell viability and a high cell concentration. This protocol can also be modified to prepare a single-cell suspension from the intestines of other fish species. This research provides an efficient reference protocol and reduces the need for additional trials in the preparation of single-cell suspensions for aquaculture fish species.

This protocol describes the preparation of a high-quality single-cell suspension of Nile tilapia intestine for single-cell sequencing (Figure 1). This research shows that the collagenase/dispase mix has a good dissociation effect and is mild for intestine tissue. The selection of the optimal digestion enzyme is essential for preparing a high-quality single-cell suspension. In the preliminary work, the dissociation efficiencies of several commonly used enzymes were compared, and the results a...

Watch this Scientific Journal Video about Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing at JoVE.com

Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing

Here, we demonstrate the preparation of a high-quality single-cell suspension of tilapia intestine for single-cell sequencing.

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The ...

This protocol provides a reliable reference for preparing a high-quality single cell suspension from the Nile tilapia intestine to facilitate single-cell level studies in aquaculture fish species. The technology is highly efficient and can be implemented in most laboratory conditions. It reduces the need for additional trials to prepare single-cell suspensions for aquaculture fish species.Begin by rinsing six month old Nile tilapia fish in well aerated sterile water for 15 minutes to wash off loosely bound bacteria from the surface. For preparing the enzymatic cell dissociation reagent, dilute the collagenase dispase in PBS to a final concentration of one milligram per milliliter. Mix 95%volume of the prepared enzymatic solution and 5%volume of FBS.Then pre-cooled the centrifuge to four degree Celsius before use. For tissue dissection, place the euthanized fish on ice and collect three to four centimeters of the intestines midsection. Excise the fat and mesentery using forceps.Remove the fat attached to the intestinal surface and the elastic and malleable clear mucosa. Gently rinse the intestine fragment with sterile ice cold PBS using a syringe to wash off the intestinal contents and mucus. After several washes, dissect the fragment into small pieces and transfer them to a 1.5 milliliter tube containing one milliliter ice cold PBS.Centrifuge the mixture at 300 x g in four degrees Celsius for five minutes. And replace the supernatant with one milliliter of ice cold 0.08%BSA-DPBS. Using a glass pipette gently aspirate to resuspend the tissue fragments.After washing the pieces one more time, remove the supernatant and add one milliliter of the prepared enzymatic dissociation reagent. Also mix five microliters of RNAs inhibitor with the dissociation reagent for single-cell RNA sequencing. Place the tube at 37 degrees Celsius tempered water bath and manually invert the tube every five minutes during the 30 to 60 minutes incubation time.Once the tubes are spinned, remove the enzymatic cell dissociation reagent using wide-bore tips. Add one milliliter of ice cold 0.08%BSA-DPBS and gently pipette the cells up and down to prevent cell disruption. After centrifusion and resuspending the cells one more time, place the 40 micrometer cell strainer pre-wetted with 0.08%BSA-DPBS onto a 50 milliliter conical tube.Pass the cell suspension through the strainer. Tap the strainer, then rinse it with 200 microliters DPBS and collect the suspension at the bottom of the tube. After transferring the suspension into a 1.5 milliliter tube, add five microliters of DNAs and incubate the suspension for 15 minutes at 15 to 20 degrees Celsius.Once centrifuged, remove the supernatant and resuspend the cell pellet in 400 microliters of ice cold DPBS without calcium and magnesium ions. Then gently pipette up and down with wide-bore tips to mix the cells and repeat this process one more time. For staining, mix the cell suspension in 0.4%trypan blue solution in an equal ratio and incubate for three minutes at room temperature.Then add 10 microliters of the mixture on the glass slide. Count the viable and dead cells under the microscope in three fields and calculate the cell viability percentage. The dissociation efficiencies of several commonly used enzymes were compared.The collagenase dispase mix was verified to have a better dissociation effect than the collagenase dispase or trypsin alone and several other enzyme mixtures. The microscopic examination showed that the intestinal cells had high viability and dispersed as single cells. Most cells were viable, whereas a few were stained dead because of the permeablized cell membrane.Pay attention to the type of PBS used. Enzymatic digestion solution cannot be used with calcium/magnesium free PBS as the enzyme requires calcium/magnesium ions to function effectively. The procedure facilitates single-cell level studies in aquaculture fish species providing a valuable reference for developing cell dissociation protocols of aquaculture fish species.

single-cell-suspension-preparation-from-nile-tilapia-intestine-for

Research

JoVE Journal

Biology

Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual dissociations the gentleMACS Dissociator allows one to dissociate tissue very efficiently under controlled and reproducible conditions.  The gentleMACS Dissociator can optimally dissociate mouse spleen, combining timesaving and standardization with user-safety.

This video describes how to dissociate mouse spleens using the gentleMACS Dissociator, an automated bench-top device that can mechanically disrupt tissues using special tubes to produce viable cell suspensions. Following dissociation, spleens are filtered, centrifuged, and resuspended for further applications.

This video describes a simple, time-saving technique for automated tissue dissociation using the gentleMACS Dissociator to prepare single-cell suspensions of mouse splenocytes.

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual ...

Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of dissociating whole organs requires specific parameters in order to obtain a high number of viable cells in a reproducible manner. The gentleMACS Dissociator optimizes this task with a simple, practical protocol. The instrument contains pre-programmed settings that are optimized for the efficient but gentle dissociation of a variety of tissue types, including mouse lungs. In this publication the use of the gentleMACS Dissociator on lung tissue derived from mice is demonstrated.

Dissociating cells from specific tissue types requires specific parameters for tissue agitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on lung tissue is explained.

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of ...

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Isolation and Transcriptome Analysis of Plant Cell Types

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, which is known as cellular heterogeneity. In recent years, single-cell and cell-type isolation combined with next-generation sequencing (NGS) techniques have become important tools for studying biological processes at single-cell resolution. However, isolating plant cells is relatively more difficult due to the presence of plant cell walls, which limits the application of single-cell approaches in plants. This protocol describes a robust procedure for fluorescence-activated cell sorting (FACS)-based single-cell and cell-type isolation with plant cells, which is suitable for downstream multi-omics analysis and other studies. Using Arabidopsis root fluorescent marker lines, we demonstrate how particular cell types, such as xylem-pole pericycle cells, lateral root initial cells, lateral root cap cells, cortex cells, and endodermal cells, are isolated. Furthermore, an effective downstream transcriptome analysis method using Smart-seq2 is also provided. The cell isolation method and transcriptome analysis techniques can be adapted to other cell types and plant species and have broad application potential in plant science.

The feasibility and effectiveness of high-throughput scRNA-seq methods herald a single-cell era in plant research. Presented here is a robust and complete procedure for isolating specific Arabidopsis thaliana root cell types and subsequent transcriptome library construction and analysis.

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, ...

Two-Step Cell Digestion of Mouse Carotid Artery

Preparation of a Single-Cell Suspension from Mouse Carotid Arteries for Single-Cell Sequencing

Carotid arteries are major blood vessels in the neck that supply blood and oxygen to the brain, but carotid stenosis occurs when carotid arteries are clogged by plaque. Revealing the cellular composition of the carotid artery at the single-cell level is essential for treating carotid atherosclerosis. However, there is no ready-to-use protocol for the preparation of single-cell suspensions from carotid arteries. To obtain a suitable protocol for the dissociation of normal carotid arteries at the single-cell level with less damage to cells, we designed a two-step digestion method by integrating the digestion process of collagenase/DNase and trypsin. Acridine orange/propidium iodide (AO/PI) dual-fluorescence counting was used to detect cell viability and concentration, and it was found that the single-cell suspension satisfied the requirements for single-cell sequencing, with the viability of cells over 85% and a high cell concentration. After single-cell data processing, a median of ~2500 transcripts per cell were detected in each carotid artery cell. Notably, a variety of cell types of the normal carotid artery, including vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells (ECs), and macrophages and dendritic cells (M&#966;/DCs), were concurrently detectable. This protocol may be applied to prepare a single-cell suspension of blood vessels from other tissues with appropriate modifications.

Author Spotlight: Vascular Tissue Dissociation and Exploring Single-Cell Subclusters for Targeted Therapy 

Here we describe a two-step cell digestion protocol for preparing a single-cell suspension of mouse carotid arteries.

Carotid arteries are major blood vessels in the neck that supply blood and oxygen to the brain, but carotid stenosis occurs when carotid arteries are ...

Isolation and Digestion of Mouse Carotid Artery for Single-Cell Suspension Preparation

Transcriptome Profiling for High-Throughput Drug Screening

Cost-Efficient Transcriptomic-Based Drug Screening

Transcriptomics allows&#160;to obtain comprehensive insights into cellular programs and their responses to perturbations. Despite a significant decrease in the costs of library production and sequencing in the last decade, applying these technologies at the scale necessary for drug screening remains prohibitively expensive, obstructing the immense potential of these methods. Our study presents a cost-effective system for transcriptome-based drug screening, combining miniaturized perturbation cultures with mini-bulk transcriptomics. The optimized mini-bulk protocol provides informative biological signals at cost-effective sequencing depth, enabling extensive screening of known drugs and new molecules. Depending on the chosen treatment and incubation time, this protocol will result in sequencing libraries within approximately 2 days. Due to several stopping points within this protocol, the library preparation, as well as the sequencing, can be performed time-independently. Processing simultaneously a high number of samples is possible; measurement of up to 384 samples was tested without loss of data quality. There are also no known limitations to the number of conditions and/or drugs, despite considering variability in optimal drug incubation times.

Author Spotlight: Cost-Effective Transcriptomic Drug Screening - Unlocking New Targets 

This protocol describes a workflow from ex vivo or in vitro cell cultures to transcriptomic data pre-processing for cost-effective transcriptome-based drug screening.

Transcriptomics allows&#160;to obtain comprehensive insights into cellular programs and their responses to perturbations. Despite a significant decrease ...

Purification of Nuclei from Mouse Brain for Single-Cell Multiome Analysis

To begin, fill a glass Dounce with two milliliters of ice-cold nuclei lysis buffer containing 0.01%digitonin, and add the dissected mouse brain tissue pieces into it. Using a glass Dounce tissue homogenizer, homogenize the tissue 25 times each with pestles A and B, and transfer the resulting homogenate into a 15-milliliter tube. Add two milliliters of ice-cold nuclei lysis buffer containing 0.01%digitonin to the homogenate and incubate on ice for five minutes.Then, centrifuge the nuclei at 500 G for five minutes at four degrees Celsius. Using a micropipette, remove the supernatant and add four milliliters of ice-cold nuclei lysis buffer containing 0.01%digitonin. Filter the nuclei suspension through a 40-micrometer cell strainer.Centrifuge the nuclei again, remove the supernatant and add four milliliters of staining buffer to wash the nuclei. After filtering the suspension through a 40-micrometer cell strainer, pellet the nuclei by centrifugation and resuspend the nuclei in one milliliter of PBS containing 0.04%BSA and RNase inhibitors. To count the cells, mix 10 microliters of 0.4%trypan blue with 10 microliters of the nuclei in a 0.5-milliliter tube.Count the nuclei using an automated cell counter. Transfer 100 microliters of the nuclei to a FACS tube for the unstained control. Add 10 microliters of 7-AAD to the remaining nuclei and incubate for five minutes at four degrees Celsius, and proceed with nuclei sorting using FACS.Then, transfer 10 microliters of the sorted nuclei into a new FACS tube containing 90 microliters of DPBS with 2%heat-inactivated FBS. After centrifuging the sorted nuclei, remove the supernatant using a micropipette and resuspend the nuclei in 100 microliters of diluted nuclei buffer. Before sorting, the sample contains substantial debris with over 99%of singlets positive for nuclear stain, indicating effective cell lysis.Brain nuclei after sorting demonstrated an increase in purity from the initial 36%to nearly 100%

Integrating Single-Cell Transcriptomics with Human Intestinal Organoids

Using Human Intestinal Organoids to Understand the Small Intestine Epithelium at the Single Cell Transcriptional Level

Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid cultures provide ex vivo model systems that bridge the gap between animal models and clinical studies. The application of single cell transcriptomics to human intestinal organoid (HIO) models is revealing previously unrecognized cell biology, biochemistry, and physiology of the GI tract. The advanced single cell transcriptomics platforms use microfluidic partitioning and barcoding to generate cDNA libraries. These barcoded cDNAs can be easily sequenced by next generation sequencing platforms and used by various visualization tools to generate maps. Here, we describe methods to culture and differentiate human small intestinal HIOs in different formats and procedures for isolating viable cells from these formats that are suitable for use in single-cell transcriptional profiling platforms. These protocols and procedures facilitate the use of small intestinal HIOs to obtain an increased understanding of the cellular response of human intestinal epithelium at the transcriptional level in the context of a variety of different environments.

Author Spotlight: Integrating Single-Cell Transcriptomics with Organoid Cultures for Advanced Research and Therapeutic Insights

The protocol combines human intestinal organoid technology with single cell transcriptomic analysis to provide significant insight into previously unexplored intestinal biology.

Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid ...