isolation of single nuclei from frozen mammalian tissues for single nuclei rna sequencing

Isolamento e profilazione di singoli nuclei da tessuti di mammiferi congelati

Single-cell (sc) and single-nucleus (sn) RNA sequencing have become commonly used protocols in molecular and cellular biology due to the increased resolution of gene expression compared to bulk RNA sequencing. However, the isolation of good quality single cell and single nuclei preparations from solid tissues remains a challenge and is often the rate-limiting step in sc/sn-RNAseq experiments. Indeed, a plethora of protocols has been developed that use various chemical and mechanical procedures to obtain cell/nuclei suspensions1,2,3,4,5,6,7,8,9,10,11,12,13,14,15. Furthermore, strategies to clean up such preparations from debris/clumps, etc., range from flow sorting to filtration to washing. Such protocols are often manual (leading to user-related variability), can be time-consuming (leading to reduced cell/nucleus viability), and/or may require access to a flow cytometer for cell/nucleus sorting. This&#160;study focused on developing a simple, quick, and partially automated single nuclei isolation protocol from frozen mammalian tissues for downstream RNA sequencing applications. We focused specifically on nuclei isolation as opposed to cell isolation as it is compatible with the use of frozen tissues, rendering sample collection/processing more practical and enabling unbiased batching of samples, especially in time-course experiments. Furthermore, although the nuclear transcriptome does not fully reflect the cellular transcriptome, several studies have now shown that single nuclei RNA sequencing data is comparable to single cell RNA sequencing data for cell-type identification, even though the proportions of cell types can vary6,16,17,18,19.
Nuclei isolation consists of several steps: 1) mechanical or chemical disruption of the tissue to release the nuclei, 2) clean-up of debris and clumps, and 3) accurate counting of nuclei for preparation for downstream applications. In a number of protocols, step 1 frequently involves the use of a Dounce homogenizer in order to disrupt the tissue3,20. Alternatively, chemical methods can be used, although these often need to be optimized for different tissues2,5,6. We have experienced that a manual tissue disruption procedure is prone to operator-associated variability, leading to variable quality and yield of nuclei. In order to minimize technical variability and to have a more consistent and reproducible protocol that works across tissues, a protocol that uses a commercially available robotic tissue dissociator was developed21. For step 2, although buffer exchange is usually the simplest means for washing nuclei, we adopted the use of a relatively short sucrose gradient centrifugation step to have a more thorough removal of debris. For brain tissue specifically, we use a silica colloid gradient instead of a sucrose gradient&#160;for more effective myelin removal. Finally, for counting, the use of a hemocytometer is the gold standard for counting and visually inspecting the nuclei. In our protocol, this step can be reliably automated using a commercially available automated fluorescent cell counter22. This protocol has been tested and is compatible with several frozen mammalian tissues, including brain, kidney, spleen, and liver, from different mammalian species (rat, mouse, and non-human primate) and provides good quality nuclei for downstream single nuclei RNA sequencing with a droplet-based commercial platform. The protocol takes approximately 75 min from tissue preparation to the start of the single nuclei RNA sequencing workflow.

Autore in primo piano: Migliorare la scoperta di farmaci - Sviluppo di protocolli automatizzati e standardizzati per l'estrazione di nuclei da tessuti congelati 

Un protocollo semplice, rapido e parzialmente automatizzato per l'isolamento di singoli nuclei da tessuti di mammiferi congelati per il sequenziamento di singoli nuclei

This research focuses on supporting drug discovery studies using single nuclei RNA sequencing to investigate the efficacy and safety of potential treatments. Therefore, we are developing standardized nuclei extraction protocols for fresh frozen tissue samples from various organs. Our goal was to automate and standardize nuclei extraction to achieve consistent and reliable results by reducing non-biological variability.The advantage is the partial automation through the use of a robotic dissociator and the simple and short workflow that can be applied on various tissue samples of different species. Having standardized protocols can help us answer questions about drug biodistribution, as well as explore the mechanisms underlying any safety signals that we might observe. In the future, we will be testing the applicability of our protocol for other downstream applications, such as ATAC-seq or multiomics.We will also try to establish protocols to detect virally expressed transgenes in single nuclei.

Watch this Scientific Journal Video about Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nuclei RNA Sequencing at JoVE.com

Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nuclei RNA Sequencing  

Isolamento di singoli nuclei da tessuti di mammiferi congelati per il sequenziamento dell'RNA a nucleo singolo

Per iniziare, avviare il dissociatore robotico. Attiva il raffreddamento impostando il cursore nella parte superiore destra dello schermo su Raffredda e facendo clic su di esso per avviare il raffreddamento in modo che il dispositivo di scorrimento appaia arancione. Successivamente, verificare che i flaconi NSR e NIB allegati abbiano liquido sufficiente e siano adeguatamente raffreddati.Rimuovere i campioni di tessuto dal congelatore a meno 80 gradi Celsius e metterli immediatamente sul ghiaccio secco. Usando un bisturi preraffreddato, tagliare il tessuto in un pezzo da 15 a 50 milligrammi su una piastra di Petri preraffreddata, o piastra metallica, posta su ghiaccio secco. Rimuovere la cartuccia di isolamento dei nuclei dal frigorifero e disimballarla.Rimuovere il macinino e pipettare 15 microlitri di inibitore dell'RNA sul fondo della cartuccia. Usando una pinzetta, posizionare il campione di tessuto sul fondo della cartuccia. Quindi, seleziona Esegui un protocollo sullo strumento e fai clic sull'opzione Nuclei nell'angolo in alto a sinistra.Dal menu, selezionare il protocollo di isolamento Low Volume Nuclei, quindi fare clic su Modifica per verificare che la velocità di interruzione sia impostata su più veloce. Quindi, aprire lo sportello dello strumento. Inserire la cartuccia nella posizione designata.Ruotare il blocco della cartuccia e farlo scorrere nel stage finché la manopola rossa non scatta in posizione. Chiudere lo sportello e fare clic su Avanti per avviare l'estrazione dei nuclei. Una volta terminata la corsa, rimuovere la cartuccia dallo strumento sollevando la manopola rossa ed estraendo il tavolino.Posizionare immediatamente la cartuccia sul ghiaccio.

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Research

JoVE Journal

Biology

Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nuclei RNA Sequencing

478 Views.  Roche Innovation Centre Basel. Per iniziare, avviare il dissociatore robotico. Attiva il raffreddamento impostando il cursore nella parte superiore destra dello schermo su Raffredda e facendo clic su di esso per avviare il raffreddamento in modo che il dispositivo di scorrimento appaia arancione. Successivamente, verificare che i flaconi NSR e NIB allegati abbiano liquido sufficiente e siano adeguatamente raffreddati.Rimuovere i campioni di tessuto dal congelatore a meno 80 gradi Celsius e metterli immediatamente su...

Video: Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nuclei RNA Sequencing  

A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they provide. Single nucleus RNA sequencing, particularly, is useful for investigating gene expression in difficult-to-dissociate tissues. Furthermore, this approach is also compatible with frozen (archival) material. Here, we describe a protocol to isolate high-quality single nuclei from frozen mammalian tissues for downstream single nucleus RNA sequencing in a partially-automated manner using commercially available instruments and reagents. Specifically, a robotic dissociator is used to automate and standardize tissue homogenization, followed by an optimized chemical gradient to filter the nuclei. Lastly, we accurately and automatically count the nuclei using an automated fluorescent cell counter. The performance of this protocol is demonstrated on mouse brain, rat kidney, and cynomolgus liver and spleen tissue. This protocol is straightforward, rapid, and readily adaptable to various mammalian tissues without requiring extensive optimization and provides good quality nuclei for downstream single nuclei RNA sequencing.

The study describes a simple, quick, and partially automated protocol to isolate high-quality nuclei from frozen mammalian tissues for downstream single nuclei RNA sequencing.

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they ...

Ricerca

Biologia

To begin, start the robotic dissociator. Turn on the cooling by setting the slider at the top of the right of the screen to Cool, and clicking on it to start cooling so that the slider appears orange. Next, check that the attached NSR and NIB bottles have sufficient liquid and are properly cooled.Remove the tissue samples from the minus 80 degrees Celsius freezer, and immediately place them on dry ice. Using a pre-cooled scalpel, cut the tissue into a 15 to 50 milligram piece on a pre-cooled Petri dish, or metal plate, placed on dry ice. Remove the nuclei isolation cartridge from the refrigerator and unpack it.Remove the grinder, and pipette 15 microliters of RNAs inhibitor to the bottom of the cartridge. Using tweezers, place the tissue sample at the bottom of the cartridge. Next, select Run a Protocol on the instrument, and click on the Nuclei option in the upper left corner.From the menu, select the Low Volume Nuclei isolation protocol, and then click on Modify to verify that the disruption speed is set to fastest. Then, open the instrument door. Insert the cartridge into the designated location.Rotate the cartridge lock, and slide into the stage until the red knob clicks into place. Close the door and click Next to start the nuclei extraction run. Once the run is finished, remove the cartridge from the instrument by lifting the red knob and pulling out the stage.Immediately place the cartridge on ice.

Clean-Up and Counting of Isolated Single Nuclei from Frozen Mammalian Tissues

After isolating single nuclei from frozen mammalian tissues, carefully pierce the round foil on the dissociator cartridge using a pipette tip. To facilitate sucrose gradient cleanup, remove 900 microliters of the nuclei suspension from the cartridge and add it to 500 microliters of sucrose cushion solution or SCS prepared in a two milliliter tube. Mix well by pipetting until the mixture is homogenous.Next, hold the tube at an angle, and carefully add 1, 400 microliters of nuclei suspension SCS mixture drop-wise onto a new 500 microliters of SCS, creating a clearly visible phase separation. Carefully close the tube, and place it back on the ice without disturbing the phase separation. Carefully spin the tubes at 13, 000g for 15 minutes at four degrees Celsius in a pre-cooled centrifuge.Remove the supernatant without disturbing the palette, and carefully resuspend the palette in 50 microliters of ice-cold NSR. Pull the two palettes of the same sample in a new 1.5 milliliter tube. Add 900 microliters of ice-cold NSR to a total volume of one milliliter, and mix well by pipetting.Centrifuge the sample at 500g for five minutes at four degrees Celsius with a swinging bucket rotor centrifuge. Remove the supernatant and resuspend the pellet in 100 microliters of PBS. For counting, dilute 10 microliters of nuclei suspension in 20 microliters of PBS.Add 25 microliters of propidium iodide staining solution to a mixing well of the fluorescent counter counting plate. Then add 25 microliters of diluted nuclei suspension, and mix well by pipetting. Transfer 50 microliters of the stained sample from the mixing well to the loading well.Load the counting plate on the cell counter and start the count. After counting, dilute the samples with PBS to the desired concentration for single nuclei RNA sequencing. Using the partially automated nuclei isolation protocol, good quality nuclei were obtained without signs of blabbing, debris or clumping.Violin plots representing the distribution of gene counts, UMI counts and mitochondrial content in each sample are shown. Expected cell types from each tissue were identified, and all animals contributed to all clusters, indicating low technical variability introduced by the protocol. Furthermore, cellular proportions, UMI counts and gene counts were comparable across all three samples per tissue type.