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1. Tissue Dissection and Dissociation
<ol>
	<li>Transfer fresh frozen tissue sample (10&#8211;60 mg) to a pre-chilled Petri dish. Mince/chop fresh frozen tissue with a razor blade to small pieces on ice.</li>
	<li>Add 500 &#956;L of chilled nuclei lysis buffer to a pre-chilled 1.5 mL tube. Place the tissue pieces in the 1.5 mL tube containing the nuclei lysis buffer and transfer to a Douncer (Table of Materials). 
	NOTE: There are two Dounce homogenizer pestles: &#8220;loose&#8221; or &#8220;A&#8221; pestle for initial sample reduction and &#8220;tight&#8221; or &#8220;B&#8221; pestle for complete sample homogenization.</li>
	<li value="3">Dounce the tissue pieces with the &#8220;loose&#8221; pestle for about 20 strokes, until friction is reduced. If debris is present, the sample may be pre-cleared by filtration with a 100 mm strainer.</li>
	<li>Dounce with the &#8220;tight&#8221; pestle for 20 strokes to achieve complete tissue homogenization.</li>
	<li>Transfer the homogenate (about 500 &#956;L) into a pre-chilled 2 mL tube. Add 1 mL of chilled lysis buffer. Mix gently and incubate on ice for 5 min. Gently mix with a wide-bore pipette tip and repeat 1&#8211;2 times during the incubation.</li>
	<li>Filter the entire homogenate using a 30 &#956;m strainer mesh, collect into a 15 mL Falcon tube and transfer back into a new pre-chilled 2 mL tube. A single strainer is typically sufficient for the entire homogenate.</li>
	<li>Check under a light microscope to verify the removal of large debris and the intactness of the nuclear membrane. Nuclei need to be round and the nuclear membrane should not be distorted. If debris is present, nuclei can be re-filtered.</li>
	<li>Centrifuge the nuclei at 500 x g for 5 min at 4 &#176;C on a benchtop centrifuge. Remove the supernatant, leaving behind ~50 &#956;L with a pellet containing the nuclei. Gently resuspend the pellet in another 1 mL of nuclei lysis buffer and incubate for 5 min on ice.</li>
	<li>Centrifuge the nuclei at 500 x g for 5 min at 4&#176;C. Remove the supernatant without disturbing the pellet, add 500 mL of 1x homogenization buffer (HB) (Table 1) and incubate for 5 min without resuspending. Then, resuspend the nuclei in another 1.0 mL of 1x HB.</li>
	<li>Centrifuge the nuclei at 500 x g for 5 min at 4 &#176;C. Remove the supernatant and gently resuspend the nuclei in 200 &#956;L of 1x HB into a new 2.0 mL tube.
	Table 1: Preparation of 1x Homogenization Buffer Unstable Solution (2 mL per sample)
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td colspan="3">1x Homogenization Buffer Unstable Solution (2 mL per sample)</td>
			</tr>
			<tr>
				<td>Reagent</td>
				<td>Final Conc.</td>
				<td>Vol per sample (&#956;L)</td>
			</tr>
			<tr>
				<td>6x Homogenization Buffer Unstable</td>
				<td>1x</td>
				<td>333.33</td>
			</tr>
			<tr>
				<td>1 M Sucrose</td>
				<td>320 mM</td>
				<td>640.00</td>
			</tr>
			<tr>
				<td>50 mM EDTA</td>
				<td>0.1 mM</td>
				<td>4.00</td>
			</tr>
			<tr>
				<td>10% NP40</td>
				<td>0.1%</td>
				<td>20.00</td>
			</tr>
			<tr>
				<td>H2O</td>
				<td></td>
				<td>1006.27</td>
			</tr>
		</tbody>
	</table>
	</li>
</ol>

<table><tbody><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>&amp;nbsp;M6250</td><td></td></tr><tr><td>CaCl2&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>21115-100ML</td><td></td></tr><tr><td>EDTA (0.5 M)</td><td>&amp;nbsp;Thermo Scientific&amp;nbsp;</td><td>R1021</td><td></td></tr><tr><td>Falcon 15 mL Conical Centrifuge Tubes</td><td>&amp;nbsp;Fisher Scientific</td><td>352096</td><td></td></tr><tr><td>MACS SmartStrainers (100 &amp;micro;m)&amp;nbsp;</td><td>Miltenyi Biotec</td><td>&amp;nbsp;130-098-463</td><td></td></tr><tr><td>Mg(Ac)2&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>63052-100ML</td><td></td></tr><tr><td>NP-40</td><td>&amp;nbsp;Abcam&amp;nbsp;</td><td>ab142227</td><td></td></tr><tr><td>Nuclei Isolation Kit: Nuclei EZ Prep</td><td>&amp;nbsp;Sigma&amp;nbsp;</td><td>NUC101-1KT</td><td></td></tr><tr><td>Safe lock tubes 1.5 mL&amp;nbsp;</td><td>Eppendorf&amp;nbsp;</td><td>30120086</td><td></td></tr><tr><td>Safe lock tubes 2.0 mL&amp;nbsp;</td><td>Eppendorf&amp;nbsp;</td><td>30120094</td><td></td></tr><tr><td>Sucrose&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>S0389</td><td></td></tr><tr><td>Wide Bore pipette tips (1000 &amp;micro;L)&amp;nbsp;</td><td>Themo Fisher Scientific&amp;nbsp;</td><td>2079GPK</td><td></td></tr></tbody></table>

isolation of nuclei from fresh frozen glioma tissues: a method to obtain intact nuclei from glioma tumor samples

Source: Narayanan, A. et al. <a href="https://www.jove.com/v/61542/nuclei-isolation-from-fresh-frozen-brain-tumors-for-single-nucleus">Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq.</a> J. Vis. Exp. (2020)
This video describes the protocol to isolate single-nuclei from fresh frozen glioma tissue. The isolated single-nuclei can be used as specimens for sequencing techniques such as single-nuclei RNA (snRNA) sequencing and single-nuclei Assay for Transposase-Accessible Chromatin (snATAC) sequencing to obtain deeper insights into inter- and intra-tumor heterogeneity observed in glioma of the central nervous tissues.

Isolation of Nuclei from Fresh Frozen Glioma Tissues: A Method to Obtain Intact Nuclei from Glioma Tumor Samples

Source: Narayanan, A. et al. Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq. J. Vis. Exp. (2020)
This video ...

To isolate nuclei, begin by taking a fresh frozen glioma sample - a tumor that originates in the glial cells of the brain - in a Petri dish.
Place the petri dish on ice to avoid tissue degradation.
Next, mince the tissue sample into small pieces.
Add a pre-chilled lysis buffer to the minced tissue pieces. Transfer the mixture to a Dounce&#160; homogenizer. Using the pestle, homogenize the tissue to disintegrate the cells and initiate lysis.
Now, transfer the homogenate to a microcentrifuge tube containing a fresh lysis buffer.
Mechanically disrupt the homogenate using a wide-bore pipette tip. This step facilitates the release of cytoplasmic organelles from the cells.
Subsequently, using an appropriately sized cell strainer, filter the homogenate into a fresh tube to remove tissue debris and other contaminants.
After filtration, take the filtrate containing various cell organelles.
Centrifuge the filtrate at a low speed to pellet the denser nuclei at the bottom while the other cell organelles remain in the supernatant.
Discard the organelle-containing supernatant. Store the crude nuclei-rich pellet in a suitable storage buffer for further downstream sequencing.

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1.1K 조회수. 출처: Narayanan, A. et al. 단일 핵 RNA-seq 및 ATAC-seq에 대한 신선 냉동 뇌 종양에서 핵 분리. J. Vis. 특급 (2020년) 이 비디오는 신선 동결 신경교종 조직에서 단핵을 분리하는 프로토콜에 대해 설명합니다. 분리된 단핵은 중추 신경 조직의 신경교종에서 관찰된 종양 간 및 종양 내 이질성에 대한 더 깊은 통찰력을 얻기 위해 snRNA(single-nuclei RNA) 염기서열 분석 및 snATAC(Single-nuclei Assay for Tra...

비디오: 신선 냉동 신경교종 조직에서 핵 분리: 신경교종 종양 샘플에서 온전한 핵을 얻는 방법 - 개념

A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a challenge for ChIP due to technical difficulty in isolation of high-quality nuclei with minimal contamination of myofibrils. Previous protocols have attempted to purify nuclei before cross-linking, which incurs the risk of altered DNA-protein interaction during the prolonged nuclei preparation process. In the current protocol, we first cross-linked the skeletal muscle tissue collected from mice, and the tissues were minced and sonicated. Since we found that ultracentrifugation was not able to separate nuclei from myofibrils using cross-linked muscle tissue, we devised a sequential filtration procedure to obtain high-quality nuclei devoid of significant myofibril contamination. We subsequently prepared chromatin by using an ultrasonicator, and ChIP assays with anti-BMAL1 antibody revealed robust circadian binding pattern of BMAL1 to target gene promoters. This filtration protocol constitutes an easily applicable method to isolate high-quality nuclei from cross-linked skeletal muscle tissue, allowing consistent sample processing for circadian and other time-sensitive studies. In combination with next-generation sequencing (NGS), our method can be deployed for various mechanistic and genomic studies focusing on skeletal muscle function.

We present a filtration-based protocol to isolate high-quality nuclei from cross-linked mouse skeletal muscle wherein we removed the need for ultracentrifugation, making it easily applicable. We show that chromatin prepared from the nuclei is suitable for chromatin immunoprecipitation and likely chromatin immunoprecipitation sequencing studies.

Chromatin Immunoprecipitation을위한 Cross-linked Skeletal Musculus로부터 고품질 핵을 준비하기위한 Filtration 기반 방법

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a ...

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JoVE Journal

생화학

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

대규모 병렬 단일 핵 RNA 시퀀싱에 대 한 성인 척수 핵의 절연

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

신경과학

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with&#160;intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

세제 및 효소 없는 방법을 사용하여 조직 전체에서 핵 분리

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension ...

Isolation of Nuclei from Fresh Frozen Glioma Tissues: A Method to Obtain Intact Nuclei from Glioma Tumor Samples

내레이션 대본

Isolation of Nuclei from Fresh Frozen Glioma Tissues: A Method to Obtain Intact Nuclei from Glioma Tumor Samples