تم دعم هذا البحث من قبل ERA-CVD-2019 و ANR-JCJC-2020 إلى SS. نشكر مرفق علم الجينوم والمعلوماتية الحيوية (GBiM) من مختبر U 1251 / Marseille Medical Genetics والمراجعين المجهولين على تقديم تعليقات قيمة.

عزل النوى من الخلايا السلفية القلبية للفأر بدقة خلية واحدة

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+multi-omic+characterization+of+congenital+heart+disease.">Integrated multi-omic characterization of congenital heart disease.</a> Nature. 608 (7921), 181-191 (2022).</li><li>Chen, Z., Wei, L., Duru, F., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RNA+sequencing:+In-depth+decoding+of+heart+biology+and+cardiovascular+diseases.">Single-cell RNA sequencing: In-depth decoding of heart biology and cardiovascular diseases.</a> Current Genomics. 21 (8), 585-601 (2020).</li><li>Machado, L., Relaix, F., Mourikis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+relief:+Emerging+methods+to+mitigate+dissociation-induced+artefacts.">Stress relief: Emerging methods to mitigate dissociation-induced artefacts.</a> Trends in Cell Biology. 31 (11), 888-897 (2021).</li><li>Tabula Muris, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcriptomics+of+20+mouse+organs+creates+a+Tabula+Muris.">Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris.</a> Nature. 562 (7727), 367-372 (2018).</li><li>Machado, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+damage+induces+a+conserved+stress+response+that+initiates+quiescent+muscle+stem+cell+activation.">Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation.</a> Cell Stem Cell. 28 (6), 1125-1135 (2021).</li><li>Haghverdi, L., Lun, A. T. L., Morgan, M. D., Marioni, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Batch+effects+in+single-cell+RNA-sequencing+data+are+corrected+by+matching+mutual+nearest+neighbors.">Batch effects in single-cell RNA-sequencing data are corrected by matching mutual nearest neighbors.</a> Nature Biotechnology. 36 (5), 421-427 (2018).</li><li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SMNN:+Batch+effect+correction+for+single-cell+RNA-seq+data+via+supervised+mutual+nearest+neighbor+detection.">SMNN: Batch effect correction for single-cell RNA-seq data via supervised mutual nearest neighbor detection.</a> Briefings in Bioinformatics. 22 (3), 097 (2021).</li><li>Stefanovic, S., Christoffels, V. 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ليس لدى المؤلفين ما يكشفون عنه.

يوفر تحليل التركيب الخلوي للقلب النامي من خلال دراسات snRNA-seq و snATAC-seq مجتمعة فهما أعمق لأصل أمراض القلب الخلقية26. درست العديد من مختبرات الأبحاث آثار حفظ أنسجة القلب بالتبريد على snRNA-seq27. يمكن أن يكون إجراء snRNA-seq و snATAC-seq باستخدام أنسجة تشريح دقيقة جديدة من نماذج الفئ...

من بين العيوب الخلقية ، عيوب القلب الخلقية (CHDs) هي الأكثر شيوعا ، وتحدث في حوالي 1٪ من المواليد الأحياء كلعام 1,2. يتم تحديد الطفرات الجينية في أقلية فقط من الحالات ، مما يعني أن الأسباب الأخرى ، مثل التشوهات في تنظيم الجينات ، متورطة في مسببات أمراض القلبالتاجية 2,3. تطور القلب هو عملية معقدة من أنواع الخلايا المتنوعة والمتفاعلة ، مما يجعل تحديد الطفرات السببية غير المشفرة وتأثيراتها على تنظيم الجينات أمرا صعبا. يبدأ تكوين الأعضاء في القلب بالسلف الخلوي الذي يؤدي إلى ظهور أنواع فرعية مختلفة من خلايا القلب ، بما في ذلك خلايا عضلة القلب والخلايا الليفية وخلايا القلب والشغاف 4,5. يظهر علم الجينوم أحادي الخلية كطريقة رئيسية لدراسة نمو القلب وتقييم تأثير عدم التجانس الخلوي في الصحة والمرض6. سهل تطوير طرق متعددة الأوميكس للقياس المتزامن للمعلمات المختلفة وتوسيع خطوط الأنابيب الحسابية اكتشاف أنواع الخلايا والأنواع الفرعية في القلب الطبيعي والمريض6. توضح هذه المقالة بروتوكول عزل أحادي النواة موثوق به للخلايا السلفية القلبية المجمدة التي تم الحصول عليها من أجنة الفئران المتوافقة مع snRNA-seq و snATAC-seq (بالإضافة إلى snRNA-seq و snATAC-seq مجتمعة)7،8،9.
ATAC-seq هي طريقة قوية تسمح بتحديد مناطق الكروماتين المفتوحة التنظيمية ووضع النيوكليوسومات10,11. تستخدم هذه المعلومات لاستخلاص استنتاجات حول موقع عوامل النسخ وهويتها ونشاطها. وبالتالي ، يمكن تحليل نشاط عوامل الكروماتين ، بما في ذلك أجهزة إعادة التشكيل ، وكذلك نشاط النسخ لبوليميراز الحمض النووي الريبي ، لأن الطريقة حساسة للغاية لقياس التغيرات الكمية في بنية الكروماتين 1,2. وبالتالي ، يوفر ATAC-seq نهجا قويا ومحايدا للكشف عن الآليات التي تتحكم في تنظيم النسخ في نوع معين من الخلايا. كما تم التحقق من صحة بروتوكولات ATAC-seq لقياس إمكانية الوصول إلى الكروماتين في الخلايا المفردة ، مما يكشف عن التباين في بنية الكروماتين داخل مجموعات الخلايا10،12،13.
على الرغم من حدوث تقدم ملحوظ في مجال الخلايا المفردة في السنوات الأخيرة ، إلا أن الصعوبة الرئيسية تكمن في معالجة العينات الجديدة اللازمة لإجراء هذه التجارب14. للتحايل على هذه الصعوبة ، تم إجراء اختبارات مختلفة بهدف إجراء تحليلات مثل snRNA-seq و snATAC-seq مع أنسجة أو خلايا القلب المجمدة15,16.
تم استخدام العديد من المنصات لتحليل بيانات الجينوم أحادية الخلية17. المنصات المستخدمة على نطاق واسع للتعبير الجيني أحادي الخلية وتنميط ATAC هي منصات لتغليف قطرات الموائع الدقيقةالمتعددة 17. نظرا لأن هذه المنصات تستخدم غرف الموائع الدقيقة ، يمكن للحطام أو الركام أن يسد النظام ، مما يؤدي إلى بيانات غير قابلة للاستخدام. وبالتالي ، فإن نجاح دراسات الخلية الواحدة يعتمد على العزل الدقيق للخلايا / النوى الفردية.
يستخدم البروتوكول المقدم هنا نهجا مشابها للدراسات الحديثة باستخدام snRNA-seq و snATAC-seq لفهم عيوب القلب الخلقية18،19،20،21،22،23. يستخدم هذا الإجراء التفكك الأنزيمي لأنسجة القلب التي تم تشريحها حديثا متبوعا بالحفظ بالتبريد للخلايا السلفية القلبية للفئران. بعد ذوبان الجليد ، يتم تنقية الخلايا القابلة للحياة ومعالجتها للعزل النووي. في هذا العمل ، تم استخدام هذا البروتوكول بنجاح للحصول على بيانات snRNA-seq و snATAC-seq من نفس التحضير النووي للخلايا السلفية القلبية للفأر.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2100 Bioanalyzer Instrument</td>
			<td>Agilent</td>
			<td></td>
			<td>No catalog number</td>
		</tr>
		<tr>
			<td>5M Sodium chloride (NaCl)</td>
			<td>Sigma</td>
			<td>59222C-500ML&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA 10%&#160;</td>
			<td>Sigma</td>
			<td>A1595-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium Next GEM Chip J Single Cell Kit, 16 rxns</td>
			<td>10X Genomics</td>
			<td>1000230</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle, 4 rxns (including Nuclei Buffer 20X)</td>
			<td>10X Genomics</td>
			<td>1000285</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess cell counting chamber slides</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess III FL</td>
			<td>Thermofisher</td>
			<td></td>
			<td>No catalog number</td>
		</tr>
		<tr>
			<td>Digitonin (5%)</td>
			<td>Thermofisher</td>
			<td>BN2006</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2650-5x5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA LoBind Tubes&#160;</td>
			<td>Eppendorf</td>
			<td>22431021</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Thermofisher</td>
			<td>14190094</td>
			<td>Sterile and RNase-free</td>
		</tr>
		<tr>
			<td>Dual Index Kit TT Set A 96 rxns</td>
			<td>10X Genomics</td>
			<td>1000215</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15 mL Conical Centrifuge Tubes&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 mL Conical Centrifuge Tubes&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>10788561</td>
			<td></td>
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		<tr>
			<td>HI-FBS</td>
			<td>Thermofisher</td>
			<td>A3840001</td>
			<td>Heat inactivated</td>
		</tr>
		<tr>
			<td>High sensitivity DNA kit</td>
			<td>Agilent</td>
			<td>5067-4626</td>
			<td></td>
		</tr>
		<tr>
			<td>Igepal CA-630</td>
			<td>Sigma</td>
			<td>I8896-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Viability/Cytotoxicity Kit</td>
			<td>Thermofisher</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Dead Cell Removal kit: Dead Cell Romoval MicroBeads, Binding Buffer 20X</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-101</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS SmartStrainers (30 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-458</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride (MgCl2)</td>
			<td>Sigma</td>
			<td>M1028-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Milieu McCoy 5A&#160;</td>
			<td>Thermofisher</td>
			<td>16600082</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>NovaSeq 6000 S2</td>
			<td>Illumina</td>
			<td></td>
			<td>No catalog number</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin (Pen/Strep)</td>
			<td>Thermofisher</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>PluriStrainer Mini 40&#181;m</td>
			<td>PluriSelect</td>
			<td>V-PM15-2021-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Rock inhibitor</td>
			<td>Enzo Life Sciences</td>
			<td>ALX-270-333-M005</td>
			<td></td>
		</tr>
		<tr>
			<td>Single Index Kit N Set A, 96 rxn</td>
			<td>10X Genomics</td>
			<td>1000212</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard 90mm Petri dish Sterilin</td>
			<td>Thermofisher</td>
			<td>101R20</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile double-distilled water</td>
			<td>Thermofisher</td>
			<td>R0582</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Hydrochloride solution (HCl)</td>
			<td>Sigma</td>
			<td>T2194-100ML&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue stain (0.4%)</td>
			<td>Invitrogen</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.05% - EDTA 1X</td>
			<td>Thermofisher</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Sigma</td>
			<td>P9416-50ML&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide orifice filtered pipette tips 200 &#956;l</td>
			<td>Labcon</td>
			<td>1152-965-008-9</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEISS SteREO Discovery.V8</td>
			<td>ZEISS</td>
			<td></td>
			<td>No catalog number</td>
		</tr>
	</tbody>
</table>

nuclei isolation from mouse cardiac progenitor cells for epigenome and gene expression profiling at single-cell resolution

القلب النامي هو هيكل معقد يحتوي على خلايا سلفية مختلفة تتحكم فيها آليات تنظيمية معقدة. يسمح فحص التعبير الجيني وحالة الكروماتين للخلايا الفردية بتحديد نوع الخلية وحالتها. كشفت مناهج تسلسل الخلية الواحدة عن عدد من الخصائص المهمة لعدم تجانس الخلايا السلفية القلبية. ومع ذلك ، تقتصر هذه الطرق بشكل عام على الأنسجة الطازجة ، مما يحد من الدراسات ذات الظروف التجريبية المتنوعة ، حيث يجب معالجة الأنسجة الطازجة مرة واحدة في نفس الجري لتقليل التباين التقني. لذلك ، هناك حاجة إلى إجراءات سهلة ومرنة لإنتاج البيانات من طرق مثل تسلسل الحمض النووي الريبي أحادي النواة (snRNA-seq) ومقايسة النواة المفردة للكروماتين الذي يمكن الوصول إليه بواسطة transposase مع تسلسل عالي الإنتاجية (snATAC-seq) في هذا المجال. هنا ، نقدم بروتوكولا لعزل النوى بسرعة للنواة المزدوجة أحادية النوى اللاحقة (الجمع بين snRNA-seq و snATAC-seq). تسمح هذه الطريقة بعزل النوى من العينات المجمدة للخلايا السلفية القلبية ويمكن دمجها مع المنصات التي تستخدم غرف الموائع الدقيقة.

Among birth defects, congenital heart defects (CHDs) are the most common, occurring in about 1% of live births each year1,2. Genetic mutations are identified in only a minority of cases, implying that other causes, such as abnormalities in gene regulation, are involved in the etiology of CHD2,3. Cardiac development is a complex process of diverse and interacting cell types, making the identification of causal noncoding mutations and their effects on gene regulation challenging. Organogenesis of the heart begins with cellular progenitors that give rise to different subtypes of cardiac cells, including myocardial, fibroblast, epicardial, and endocardial cells4,5.&#160;Single-cell genomics is emerging as a key method for studying heart development and assessing the impact of cellular heterogeneity in health and disease6. The development of multi-omics methods for the simultaneous measurement of different parameters and the expansion of computational pipelines has facilitated the discovery of cell types and subtypes in the normal and diseased heart6. This article describes a reliable single-nucleus isolation protocol for frozen cardiac progenitor cells obtained from mouse embryos that is compatible with downstream snRNA-seq and snATAC-seq (as well as snRNA-seq and snATAC-seq combined)7,8,9.
ATAC-seq is a robust method that allows the identification of regulatory open chromatin regions and the positioning of nucleosomes10,11. This information is used to draw conclusions about the location, identity, and activity of transcription factors. The activity of chromatin factors, including remodelers, as well as the transcriptional activity of RNA polymerase, can, thus, be analyzed since the method is highly sensitive for measuring quantitative changes in chromatin structure1,2. Thus, ATAC-seq provides a robust and impartial approach to uncovering the mechanisms controlling transcriptional regulation in a specific cell type. ATAC-seq protocols have also been validated to measure chromatin accessibility in single cells, revealing variability in the chromatin architecture within cell populations10,12,13.
Although there have been notable advances in the field of single cells in recent years, the main difficulty is the processing of the fresh samples needed to perform these experiments14. To circumvent this difficulty, various tests have been carried out with the aim of conducting analyses such as snRNA-seq and snATAC-seq with frozen cardiac tissue or cells15,16.
Several platforms have been used to analyze single-cell genomics data17. The widely used platforms for single-cell gene expression and ATAC profiling are platforms for multiple microfluidic droplet encapsulation17. As these platforms use microfluidic chambers, debris or aggregates can clog the system, resulting in non-usable data. Thus, the success of single-cell studies depends on the accurate isolation of individual cells/nuclei.
The protocol presented here uses a similar approach to recent studies using snRNA-seq and snATAC-seq to understand congenital heart defects18,19,20,21,22,23. This procedure utilizes the enzymatic dissociation of freshly microdissected cardiac tissue followed by the cryopreservation of mouse cardiac progenitor cells. After thawing, the viable cells are purified and processed for nuclear isolation. In this work, this protocol was successfully used to obtain snRNA-seq and snATAC-seq data from the same nuclear preparation of mouse cardiac progenitor cells.

تسليط الضوء على المؤلف: عزل النوى من الخلايا السلفية القلبية للفأر من أجل التنميط epigenome والتعبير الجيني بدقة خلية واحدة  

عزل النوى من الخلايا السلفية القلبية للفأر من أجل التنميط فوق الجينوم والتعبير الجيني بدقة خلية واحدة

The main objective of this protocol is to study the interaction of genetic and environmental factors in the context of heart development and their contribution to congenital heart defects. Single nuclei approaches such as single nuclei RNA-seq and single nuclei ataxic are emerging as key methods to study cellular heterogeneity in health and disease during heart development. One limiting step of these experiments is that all the sample have to be run simultaneously.While working with all the experimental conditions, collecting fresh enough material is for all the condition simultaneously could be challenging. Although there have been a significant progress in the field in single cell for recent years, the main difficulty is processing free samples. Avoiding the difficulty is extremely benefit to identify the molecular mechanism underlying congenital heart disease defects.This protocol describes the steps to follow for successfully isolating high quality nuclei from frozen cardiac cell suspension obtained from mouse embryos. And our protocol is compatible with downstream single nuclei RNA-seq and single nuclei ataxic. We hope that this procedure will help interested researchers and encourage them to use this powerful method for their research.

بالمقارنة مع إعداد معلقات أحادية الخلية لنهج الخلية الواحدة ، فإن إعداد معلقات أحادية النوى أكثر صعوبة ويتطلب درجة أعلى من الدقة والمعالجة. العامل الرئيسي لنجاح snRNA-seq و snATAC-seq هو تعليق نوى نظيف وسليم. يجب تكييف بروتوكول عزل النوى الفعال مع كل نوع من الأنسجة وحالتها (طازجة أو مجمدة). هنا ، يت?...

Watch this Scientific Journal Video about Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution at JoVE.com

هنا ، نقدم بروتوكولا يصف تحضير نوى الخلية. بعد التشريح المجهري والتفكك الأنزيمي للأنسجة القلبية إلى خلايا مفردة ، تم تجميد الخلايا السلفية ، تليها عزل الخلايا النقية القابلة للحياة ، والتي تم استخدامها لتسلسل الحمض النووي الريبي أحادي النواة ومقايسة النواة المفردة للكروماتين الذي يمكن الوصول إليه بواسطة transposase مع تحليلات تسلسل عالية الإنتاجية.

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...

الهدف الرئيسي من هذا البروتوكول هو دراسة تفاعل العوامل الوراثية والبيئية في سياق نمو القلب ومساهمتها في عيوب القلب الخلقية. تظهر مناهج النوى المفردة مثل RNA-seq أحادي النوى وترنح النوى المفردة كطرق رئيسية لدراسة عدم التجانس الخلوي في الصحة والمرض أثناء نمو القلب. إحدى الخطوات المحددة لهذه التجارب هي أنه يجب تشغيل جميع العينات في وقت واحد.أثناء العمل مع جميع الظروف التجريبية ، فإن جمع مواد جديدة كافية لجميع الحالات في وقت واحد قد يكون أمرا صعبا. على الرغم من إحراز تقدم كبير في هذا المجال في خلية واحدة خلال السنوات الأخيرة ، إلا أن الصعوبة الرئيسية تكمن في معالجة العينات المجانية. تجنب الصعوبة مفيد للغاية لتحديد الآلية الجزيئية الكامنة وراء عيوب أمراض القلب الخلقية.يصف هذا البروتوكول الخطوات التي يجب اتباعها لعزل النوى عالية الجودة بنجاح من معلق خلايا القلب المجمدة التي تم الحصول عليها من أجنة الفئران. وبروتوكولنا متوافق مع المصب أحادي النواة RNA-seq وترنح أحادي النواة. نأمل أن يساعد هذا الإجراء الباحثين المهتمين ويشجعهم على استخدام هذه الطريقة القوية لأبحاثهم.

nuclei-isolation-from-mouse-cardiac-progenitor-cells-for-epigenome

Research

JoVE Journal

Developmental Biology

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

1.4K Views.  Aix-Marseille University. الهدف الرئيسي من هذا البروتوكول هو دراسة تفاعل العوامل الوراثية والبيئية في سياق نمو القلب ومساهمتها في عيوب القلب الخلقية. تظهر مناهج النوى المفردة مثل RNA-seq أحادي النوى وترنح النوى المفردة كطرق رئيسية لدراسة عدم التجانس الخلوي في الصحة والمرض أثناء نمو القلب. إحدى الخطوا...

Video: تسليط الضوء على المؤلف: عزل النوى من الخلايا السلفية القلبية للفأر من أجل التنميط epigenome والتعبير الجيني بدقة خلية واحدة  

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses.

Mouse Cardiac Progenitor Cell Isolation, Digestion, and Freezing

Begin by placing the embryos dissected from the euthanized two to six-month-old pregnant female mouse in a cold complete medium with 1%fetal bovine serum. After removing the endometrial tissue, placenta, and yolk sack from the embryo under 5X magnification, dissect the cardiac progenitors from the embryonic heart region using forceps. Pull all the heart regions dissected from five mouse embryos in a 1.5 milliliter micro centrifuge tube and centrifuge them in a swinging bucket, pre-chilled to four degrees Celsius.Remove the supernatant, and wash the tissue with one milliliter of tissue culture grade PBS. Centrifuge the tubes at 300G for one minute at four degrees celsius. Then, remove the supernatant, and repeat two washes with one milliliter PBS.Once done, replace the PBS with 0.05%Trypsin-EDTA. Centrifuge and repeat two washes with 0.05%Trypsin-EDTA. To digest the tissue, add 50 microliters of 0.05%Trypsin-EDTA and heat for 10 minutes at 37 degrees Celsius.Next, apply a gentle mechanical dissociation after five minutes by aspirating the suspension up and down using a wide-orifice filter tip. Inactivate the Trypsin digestion activity by adding 350 microliters of complete medium to the dissociated cells. Pass the cell suspension through a 40 micrometer cell strainer.Prepare the cells for freezing by centrifuging the freshly-dissociated cell suspension. Carefully remove the supernatant and resuspend the pellet in one milliliter of chilled freezing medium. Transfer the cell suspension into a pre-cooled cryo vial, and place the cryo vial in a pre-cooled freezing container.Place the freezing container at minus 80 degrees Celsius for four to eight hours before transferring it to liquid nitrogen for sequencing experiments.

عزل الخلايا السلفية القلبية للفأر والهضم والتجميد

Embryonic Heart Cell Thawing and Removal of Dead Cells

Begin by placing the frozen dissociated embryonic heart cell suspension containing cryo vials in a 37 degrees Celsius water bath for one to two minutes. Add one milliliter of complete medium, pre warmed at 37 degrees Celsius to the thawed suspension and mix it three times with a pipette. Then transfer the suspension to a 15 milliliter conical tube, containing 10 millimeters of warm complete medium.Pass the entire cell suspension over a 30 micrometer strainer and rinse the strainer with one to two milliliters of warm, complete medium. Collect the flow through in the same conical tube. Next, centrifuge the tubes at 300 G for five minutes.After removing the supernatant, wash the pellet with PBS and transfer the cell suspension into a 1.5 milliliter micro tube. Centrifuge the cell suspension and add 100 microliters of the provided magnetic microbeads to the pelleted cells. Homogenize the suspension five times using a wide boar pipette tip before 15 minutes of incubation at room temperature.In the meantime, rinse the magnetic separation column with 500 microliters of binding buffer. Once the incubation is complete, dilute the microbead cell suspension mixture using 500 microliters of binding buffer. Next, add 0.6 milliliters of the cell suspension to the magnetic separation column.Collect the live cell effluent in a 15 milliliter centrifuge tube. Next, rinse the 1.5 milliliter micro tube that contained the cell suspension using one milliliter of binding buffer. After rinsing, transfer the binding buffer from the 1.5 milliliter micro tube to the column and collect the effluent in the same 15 milliliter tube.Repeat the rinsing of the 1.5 milliliter tube using one milliliter of binding buffer. After centrifuging the effluent at 300 G for five minutes, remove the supernatant without disrupting the cell pellet. Add one milliliter of the PBS BSA solution and gently mix by pipetting using a wide orifice pipette tip.Then transfer the cell suspension into a 1.5 milliliter micro tube. Again, centrifuge the cell suspension and remove the supernatant without disrupting the cell pellet. Repeat the two washes of one milliliter of PBS BSA.After removing the one milliliter of PBS BSA, resuspend the cells in 100 microliters of PBS BSA solution. Mix it by pipetting 10 times. Determine the concentration by performing a trypan blue assay to ensure a cell count of more than or equal to 100, 000 cells and perform a fluorescence based assessment for the viability of the cells.The additional step of sorting and removing the dead cells after thawing allowed the procurement of a cleaner cell suspension with significantly higher cell viability and improved the quality of the starting sample. For more accuracy, two different counting methods were used to quantify the cells and nuclei, including trypan blue and the fluorescence based assessment of the viability. In this protocol, it was observed that the cell viability passed from 80 to 90%before nuclei isolation to less than 5%after nuclei isolation.

ذوبان خلايا القلب الجنينية وإزالة الخلايا الميتة

Nuclei Isolation from Cardiac Progenitor Cells

To begin, clean the working place and materials with 70%ethanol and RNase removing solution. Freshly prepare lysis buffer, lysis dilution buffer, 0.1 x lysis buffer, and wash buffer and maintain the buffers on ice. Pre-chill swinging bucket centrifuges to four degrees Celsius.Centrifuge the cardiac progenitor cell suspension in a swinging bucket centrifuge. Gently remove all the supernatant without touching the bottom of the tube. Add 100 microliters of chilled 0.1 x lysis buffer to the cell suspension, and mix it by pipetting 10 times.After five minutes of incubation on ice, add one milliliter of chilled wash buffer and mix it. After centrifuging the suspension, remove the supernatant and repeat two more washes with chilled wash buffer. Prepare the diluted nuclei buffer.After resuspending the nuclei in the nuclei buffer, place the nuclei stock solution on ice. When the quality of the isolated nuclei was assessed by the evaluation of the nuclear membrane morphology, the isolated nuclei appeared smooth and uniformly round under Bright-field microscopy indicating an effective isolation process.