This work was supported in part by grants from the National Institute of Environmental Health Sciences (ES014443 and ES017274) and AstraZeneca to KSR.

NOTA: Todos os passos devem ser realizados à temperatura ambiente a menos que especificado de outra forma. Consulte a seção Reagentes para obter detalhes sobre como preparar reagentes individuais. 1. Rotulagem L1 Sondas NOTA: As sondas podem ser marcadas através de marcação química ou PCR. <ol><li> marcação química <ol><li> Adicione 0,7-1% de gel de agarose em Tris-acetato-EDTA (TAE) tampão, calor num forno de microondas até derreter, permitir que o gel arrefecer, e, em seguida adicionar brometo de etídio (0,5 ug / ml). Verter em tabuleiro de gel, adicionar pentes e para permitir que o gel solidificar à temperatura ambiente. </li><li> Rotular a sonda Sneo (1-2 ug) com CY3 ou FITC a 37 ° C durante 1 hora de acordo com o protocolo do fabricante. NOTA: o S indica a Sneo pliced ​​gene neo micina expressa após um ciclo completo de retrotransposição. A sonda é ~ 1000 bp de tamanho. Sneo pode ser amplificado por PCR a partir de qualquer vector ou de genomADN IC. Veja a Tabela de Materiais e Reagentes para obter informações sobre Estreptavidina-CY3 / reagentes de marcação FITC. </li><li> Misturar a solução de sonda Sneo marcado com tampão de carregamento de DNA 1x, a carga em cada poço e executar a 75-100 volts para 15-20 min. </li><li> Visualize a banda sonda usando um Ultra Violeta-Iluminador (UV). Note-se que o tamanho da sonda Sneo podem ser ajustados e que o bloqueio de ácido núcleos (LNA) pode também ser ser adicionado (Figura 2). </li><li> Corte sonda marcada Sneo a partir do gel com uma lâmina de limpeza, solubilizar e purifica-se a sonda Sneo a partir do gel utilizando um kit PCR Clean-Up de acordo com o protocolo do fabricante. CUIDADO: Tampa todas as partes expostas do corpo com escudos de proteção (isto é, casacos de laboratório e UV máscara clara) ao visualizar e cortando o gel. Veja a Tabela de materiais e reagentes para informação gel purificar produtos de PCR. </li><li> Re-run 5 mL de Sneo sonda marcada com uma sonda Sneo marcado com un em gel de agarose 0,7% (ver 1.1.1) para conaumento do tamanho firme e perda de intensidade de sinal (Figura 2). </li><li> Quantificar a quantidade de sonda etiquetada Sneo através da medição da absorvância a 260 nm e diluir a sonda Sneo a 10 ng / mL alíquotas em nuclease livre H 2 O. alíquotas armazenar a -20 ° C. </li></ol></li><li> PCR Labeling (método alternativo para a rotulagem de sonda) <ol><li> Combinar iniciadores específicos Sneo (5&#39;-ggatagcattgggagatatacct-3 &#39;e 5&#39;-attgaacaagatg gattgcacgc-3&#39;) com reagentes de PCR num único tubo: 13,6 ul de nuclease livre H 2 O; 0,1 ul de dATP, dCTP, dGTP (10 nM); 0,05 jil dTTP (10 nM); 0,05 ul de dUTP-biotina / dUTP-FITC / CY3 (10 nM); 4 ul Ir Tag 5x tampão; 0,4 ul Ir Tag polimerase; 1 ul Sneo molde (10 ng). Veja a Tabela de Materiais e Reagentes para obter informações sobre reagentes de PCR. </li><li> Ajustar a quantidade de cada reagente, multiplicando pelo número total de amostras. </li><li> Use o seguinte parâmetro de ciclismo PCRs para amplificar e etiqueta Sneo sondas: 95 ° C durante 2 min; 35 ciclos de 95 ° C durante 30 seg, 62 ° C durante 30 seg, 72 ° C durante 60 seg; 72 ° C durante 2 min; Manter a 4 ° C indefinidamente. NOTA: A temperatura de recozimento pode ser ajustado ou PCR gradiente pode ser concluída para determinar temperaturas de recozimento ideais para outras sondas. </li><li> Faça gel 0,7-1%, como no ponto 1.1.1, carga e visualizar a banda sonda como no ponto 1.1.4. </li><li> Corte e purificar a sonda Sneo rotulado como no ponto 1.1.5. </li><li> Re-run 5 mL de Sneo sonda marcada com sonda Sneo marcado com un para confirmar aumento do tamanho e perda de intensidade do sinal (veja a Figura 2). </li><li> Quantificar a quantidade de sonda Sneo marcado por absorvência de medição a 260 nm e diluir sonda Sneo a 10 ng / mL alíquotas em nuclease H 2 O. livre </li></ol></li></ol> 2. Preparar Spreads cromossômicas <ol><li> Gerar clones estáveis ​​expressando estrutura do vector (control) ou L1 retrotransposição competente, utilizando metodologias padrão de selecção. 12,16 Enquanto repórteres não epissómicos foram usadas para correlacionar os achados com estudos de transcrição, também poderiam ser utilizados vectores epissomais. Cultivar células que expressam de forma estável o controle ou vector L1 (1 x 10 6 células por placa de 10 cm, com ajustes no tamanho da placa feitas conforme necessário) em meio de crescimento completo. Para as células HepG2, utilizar meio RPMI-1600, 10% de FBS e 200 ug / ml de higromicina. Crescer as culturas a 70% de confluência a 37 ° C e 5% de CO 2. NOTA: A escolha do meio de crescimento é dependente do tipo de célula. Dado que os plasmídeos aqui utilizados para transportar cassetes de selecção tanto higromicina e neomicina, a selecção de clones estáveis ​​de também poderia ser feito com neomicina após selecção em higromicina, mas a quantidade de neomicina tem de ser optimizado para estabelecer os níveis de tolerância para cada tipo de célula. </li><li> Adicionar colcemida (0,4 ug / ml) ao meio de cultura e incuba-se durante 90 min para reter as células na metaphase. Se usando diferentes tipos de células para determinar a concentração óptima de colcemida e tempo de exposição (60, 90, e 120 min) para prisão da metafase óptima empiricamente. </li><li> Lavar as células 2x com 10 ml de PBS 1x Dulbecco (DPBS), trypsinize por adição de 3-5 ml de solução de tripsina a 0,25% para as células e incuba-se durante 5 minutos para separar as células. Inactivar a tripsina com uma quantidade igual de meio contendo 10% de FBS. </li><li> Centrifugar as células durante 2 min a 1000 xg, a 4 ° C, aspirar o meio a partir da pelete de células e lava-se as células com DPBS. Aspirar todos os DPBS, deixando 200 mL de DPBS para voltar a suspender as células. células assegurem são misturados bem e que todos os aglomerados estão dispersos. Use cintilação ou pipetagem suave para misturar e dispersar aglomerações e evitar vórtex. </li><li> Adicionar 5 ml de solução hipotónica (isto é, KCl a 75 mM) que é pré-aquecido a 37 ° C, gota a gota enquanto se roda o tubo de 15 ml horizontalmente e incubar a 37 ° C durante 20 min. Veja a Tabela de Materiais e Reagents para obter informações sobre como obter e fazer a solução hipotônica. </li><li> células centrifugar a 120 xg durante 5 min a 4 ° C e os passos de repetição 2,4-2,5 3x deixando aproximadamente 200 ul de solução hipotónica para re-suspender as células após cada lavagem. Certifique-se de que no final dos 3 lavagens, o sedimento é visivelmente branco e inchado. </li><li> Re-suspender o sedimento em 200 ul de solução de Carnoy fixador e fazer 1: 2, 1: 4, 1: 8, 1:16 diluições em solução de Carnoy fixador. Queda de 10 ul de cada diluição numa lâmina limpa seca de aproximadamente 1 cm acima e expor imediatamente a lado sem se espalhar para o vapor quente da água fervente por 30 segundos. Veja a Tabela de Materiais e Reagentes para obter informações sobre como fazer a solução de Carnoy fixador. </li><li> Secam-se as propagações à temperatura ambiente, mancha com 0,1 ug / ml de Hoechst-33342 por imersão durante 15-20 min em frasco Coplin e lavar 3x com DPBS. Veja a Tabela de Materiais e Reagentes para obter informações sobre como fazer a solução Hoechst-33342. </li> &lt;li&gt; Ver espalha em microscópio de fluorescência / de contraste de fase de 40x de ampliação. Depois de otimizar a preparação propagação, os spreads não precisam ser marcadas com Hoechst-33342. Ver se espalha em microscópio de contraste de fase em aumento de 10x para determinar a qualidade disseminação e separação, conforme descrito na seção Resultados (ver Figura 3). </li><li> Escolha e circundar boas para barrar com um diamante ponto do marcador no lado oposto da lâmina (isto é, livre espalhar-lado). </li><li> Loja dissemina-se a -20 ° C durante até um mês. Evitar a exposição à umidade. </li></ol> 3. Fluorescence In Situ Hybridization (FISH) <ol><li> A estabilização e a desidratação de barrar NOTA: Equilibrar cromossoma metáfase se espalha até à temperatura ambiente, se for armazenado a -20 ° C. Veja a Tabela de Materiais e Reagentes para obter informações sobre como obter e fazer reagentes. <ol><li> Incubar cromossoma metáfase espalha com 200 ul de ARNase A durante1 h a 37 ° C. </li><li> Lavagem das lâminas em tampão SSC 2x-duas vezes durante 5 min cada seguido por lavagem com solução de HCl 10 mM. </li><li> Incubar barrar com 1% de pepsina durante 10 min a 37 ° C, lavar com H 2 O desionizada e lava-se duas vezes com tampão 2 x SSC-durante 5 min cada. </li><li> Incubar barrar com 4% de paraformaldeído durante 10 minutos e lava-se em tampão SSC 2x duas vezes durante 5 min cada. </li><li> Desidratar espalhar por incubação durante 2 min em série de etanol: 70%, 80%, e 95% de etanol. </li><li> lâminas de ar seco. </li></ol></li><li> Hibridação espalha com sondas retrotransposição marcado com L1 (ou seja, Sneo) CUIDADO: Para sondas diretas marcado, manter afastado de luz em todos os momentos. Veja a Tabela de Materiais e Reagentes para obter informações sobre como obter e fazer reagentes. <ol><li> Adicione 30 ng de Sneo de tampão de hibridação, aquece-se a 72 ° C durante 10 min e arrefecer durante 2 min à temperatura ambiente. </li><li> Adicionar 30 ml de solução de sonda Sneo para each spread, cubra com lamela e selar as bordas com cimento de borracha. Garantir que não haja formação de bolhas. </li><li> Aquecer a lâmina a 72 ° C durante 5 min num bloco de aquecimento, gradualmente, baixar a temperatura para 37 ° C e incubar durante a noite numa câmara humidificada escuro a 37 ° C. </li></ol></li><li> Lavar e visualizando (ou adição de anticorpo secundário) CUIDADO: Para sondas diretas marcado, manter afastado de luz em todos os momentos. NOTA: Consulte a Tabela de Materiais e Reagentes para obter informações sobre como se preparar. Uso de um volume suficiente para cobrir toda a propagação cromossoma é recomendado. Lembre-se que o excesso de volume será perdido quando lamela é adicionado. <ol><li> Imergir as lâminas em tampão SSC 2x para remover lamelas. </li><li> Lavagem das lâminas por imersão em tampão SSC 2x-a 45 ° C durante 5 min. </li><li> Lavagem das lâminas por imersão em tampão de lavagem a 45 ° C durante 5 min, 2x. </li><li> Lavagem das lâminas por imersão em tampão SSC 0,1 x a 45 ° C durante 10 min. </li><li>Lavagem das lâminas por imersão em 2X tampão SSC a 45 ° C durante 10 min. NOTA: Coloque um frasco Coplin contendo tampão recomendado num banho de água, ajustar a temperatura a 45 ° C. </li><li> Frescos lâminas à temperatura ambiente e equilibrar as lâminas em tampão de detecção. </li><li> Para sondas marcadas diretos, pule para o passo 3.4.10. </li><li> Para sondas marcadas indirectos, bloco em tampão de bloqueio durante 20-30 min e lava-se 3x em DPBS. </li><li> Incubar com 50 uL de anticorpo secundário (por exemplo, 5 ug / ml de estreptavidina-FITC, ou αCY3 em tampão de bloqueio) durante 1 h. </li><li> Lavar as lâminas em 2x SSC durante 5 minutos duas vezes. </li><li> Contracoloração com Hoechst-33342 solução (0,1 ug / ml) durante 10 min. NOTA: Esta coloração é feito para manchar cromossomos para co-localização com a sonda. </li><li> Lavar em DPBS 3x, adicione uma gota de meio de montagem, coloque uma lamela e selar as bordas com unha polonês. </li><li> Analisar L1 retrotransposição usando microscópio de fluorescência em 40X magnification. </li></ol></li></ol>

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Metodologias como toda a sequenciação do genoma, PCR inversa e transferência de Southern foram utilizados para estudar L1 retrotransposição. Embora estas metodologias são extremamente valiosas na localização onde ocorrem inserções de L1 dentro de genomas, um desafio de confusão para todos eles, é a necessidade de programação in silico para montar sequências. A metodologia aqui descrita FISH foi concebido para complementar estes métodos, especialmente no caso dos estudo...

Humano longo Intercaladas Nuclear Elemento-1 (Linha-1 ou L1) é um elemento móvel autônomo que se propaga dentro do genoma através de um &quot;copiar e colar&quot; mecanismo de retrotransposição. Um L1 humano típico é ~ 6 kb de comprimento e é composto por uma 5&#39;UTR (região não traduzida) que serve como um promotor, duas grelhas de leitura abertas (ORFs): L1 e L1 -ORF1 -ORF2, e um 3&#39;UTR com um poliA . -ORF1 cauda proteína L1 tem três domínios distintos: um domínio de bobina da bobina, ARN reconhecimento do motivo e domínio C-terminal, enquanto que a proteína L1 -ORF2 tem endonuclease, transcriptase reversa e rico em cisteína domínios 1,2,3,4,5. L1 -ORF1 exibe actividades chaperonas de ácido nucleico, enquanto L1 -ORF2 fornece actividades enzimáticas, com ambas as proteínas necessárias para a retrotransposição 1,2,6. Um ciclo de L1 retrotransposição começa com a transcrição da 5&#39;UTR de L1 </em&gt; ARNm bicistrónico pela ARN-polimerase II, a translocação para o citoplasma, e a tradução. No citoplasma, L1 -ORF1 exposições quer ligaï¿½o ao cis em que empacota o seu próprio RNA ou trans onde ligaï¿½o ao empacota outros ARN (isto é, forma de seno / Svas / pseudogenes) para formar uma partícula ribonucleoproteica (RNP) com L1 -ORF2p 7, 8. RNP translocar para o núcleo, onde a actividade de endonuclease de L1 -ORF2p cortes de ADN genómico (ADNg) para expor um OH - Grupo 9, que por sua vez é usado por transcriptase reversa para escorvar e reverso de ARN a sintetizar ADN a partir da extremidade 3 &#39;. Durante a síntese inverso, a segunda cadeia de ADN é cortado 7-20 pb a partir do site original nick para criar quebras escalonados que são preenchidos para formar a assinatura sequências de inserção L1 (por exemplo, TTTTAA) chamados duplicações no local alvo (TSD) 9,10. Este processo é conhecido como o principal alvo da transcrição reversa (TPRT) e leva a insertina de cópias completas ou truncadas de L1 / outros ADNs com TSDs em ambas as extremidades da sequência inserida. L1 retrotransposição também tem sido mostrado para ser mediada através TPRT semelhante de extremidades não homólogas-se juntar, em alguns tipos de células 11. O vector de L1 retrotransposição empregue nos nossos estudos é não-epissomal e consiste em etiquetado L1 -ORF1 &amp; 2p impulsionado por promotores CMV-L1-5&#39;UTR combinadas (Figura 1) 12. As versões anteriores desta construção foram descritos em estudos utilizando leveduras e culturas de células humanas 13,14,15. Dois promotores de CMV distintas localizadas nas extremidades 5 &#39;e 3&#39; do vector, com a extremidade 3 &#39;colocado na orientação inversa, para dirigir a expressão da neomicina após splicing e integração. A cassete de indicador retrotransposição na extremidade 3 &#39;de um composto anti-sentido do gene inserido a neomicina L1 -ORFs e tornada inactiva por separação em duas metades por uma globno intrão com splicing dador (SD) e locais de splicing aceitador (SA) (Figura 1). Após a integração num cromossoma, L1 é transcrito a partir do promotor comum para produzir um ARNm que consiste de um ARNm bicistrónico e ARNm neomicina inactivo. Durante o processamento de ARN, o intrão de globina é spliced ​​out do gene da neomicina para restaurar um gene da neomicina totalmente funcional. O ARNm híbrido é embalada em uma RNP em cis e translocado para o núcleo, onde ele é integrado no genoma como qualquer um de comprimento completo ou truncadas, utilizando inserções TPRT. Aqui nós descrevemos uma metodologia que utiliza hibridização fluorescente in situ (FISH) com sondas especificamente dirigidas ao gene neomicina emendado (Sneo) para rastrear padrões de -retrotransposiition L1 e as taxas de inserção no nível de núcleo único. A eficiência e especificidade da detecção foi confirmada usando retrotransposição competente e construções incompetentes e sondas para detect Sneo ou a neomicina e globina junção intron 16. Esta metodologia é responsável por algumas das deficiências de ensaios retrotransposição base de cultura de células, tais como várias inserções, resistência à colónia e expansão clone favorável.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Labeling Probes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Go Tag DNA polymerase</td>
			<td>Promega</td>
			<td>M3178</td>
			<td></td>
		</tr>
		<tr>
			<td>GO Tag 10X colorless buffer</td>
			<td>Promega</td>
			<td>M3178</td>
			<td></td>
		</tr>
		<tr>
			<td>Individual dNTP</td>
			<td>Sigma</td>
			<td>DNTP10</td>
			<td>Adjust the concentration of dATP, dGTP, dCTP to 2mM and dTTP to 1.5mM&#160; in nuclease free water.</td>
		</tr>
		<tr>
			<td>&#160;dUTP-16-Biotin (0.5mM)</td>
			<td>Roche</td>
			<td>11093070910</td>
			<td></td>
		</tr>
		<tr>
			<td>dUTP-FITC (0.5mM)</td>
			<td>Thermo Scientific</td>
			<td>R0101</td>
			<td></td>
		</tr>
		<tr>
			<td>dUTP-CY3 (0.5mM)</td>
			<td>Sigma</td>
			<td>GEPA53022</td>
			<td></td>
		</tr>
		<tr>
			<td>MIRUS FISH Labeling Kit</td>
			<td>MIRUS</td>
			<td>MIR3625</td>
			<td></td>
		</tr>
		<tr>
			<td>MIRUS FISH Labeling Kit</td>
			<td>MIRUS</td>
			<td>MIR3225</td>
			<td></td>
		</tr>
		<tr>
			<td>NucleoSpin PCR Clean-Up kit&#160;</td>
			<td>Qiagen</td>
			<td>1410/0030</td>
			<td>Any PCR clean reagent can be used in place of NucleoSpin PCR Clean Up Kit</td>
		</tr>
		<tr>
			<td>PCR Machine</td>
			<td></td>
			<td></td>
			<td>Any Thermocycler machine can be used to amplify the label product.</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Preparing chromosome Spreads.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; Colcemid&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>15212-012</td>
			<td>Add Colcemid directly to growth media&#160; to a final concentration of 0.4 &#181;g/mL</td>
		</tr>
		<tr>
			<td>1M KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td>Dilute 1M KCl solution to 75 mM solution to make Hypotonic Solution</td>
		</tr>
		<tr>
			<td>Carnoy Fixative Solution</td>
			<td></td>
			<td></td>
			<td>Mix 3 volumes of methanol and 1 volume of acetic acid to make Carnoy Fixative Solution. Make Fresh everytime.</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma</td>
			<td>322415</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma</td>
			<td>320099</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst-33342</td>
			<td>Thermo Scientific</td>
			<td>62249</td>
			<td>Dissolve Hoechst-33342 in PBS to final concentration of 1 mg/mL.</td>
		</tr>
		<tr>
			<td>Diamond Point Marker</td>
			<td>Thermo Scientific</td>
			<td>750</td>
			<td></td>
		</tr>
		<tr>
			<td>Frosted Slides</td>
			<td>Thermo Scientific</td>
			<td>2951-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Life Technologies&#160;</td>
			<td>R001100</td>
			<td>To detech cells, incubate cell in Trypsin for 5 mins and inactive with equal volume of complete media</td>
		</tr>
		<tr>
			<td>Cover slides&#160;</td>
			<td>VWR</td>
			<td>48366067</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin Jars</td>
			<td>Thermo Scientific</td>
			<td>107</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Heat Block</td>
			<td></td>
			<td></td>
			<td>Any heat block can be used for this purpose, though blocks fitted for heating slides are recommended.</td>
		</tr>
		<tr>
			<td>&#160;Beaker (600ml)</td>
			<td>Sigma</td>
			<td>CLS1003600</td>
			<td>Fill the beaker with water, heat to boil, use the hot steam to burst chromosomes.</td>
		</tr>
		<tr>
			<td>Nikon Microscope</td>
			<td>Nikon</td>
			<td>125690</td>
			<td>Any microscope can be used to look at spread quality.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Reagents and Materials for FISH</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trisodium citrate</td>
			<td>Sigma</td>
			<td>S1804</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chroride</td>
			<td>Sigma</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Sigma</td>
			<td>F9037</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>F1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Detran Sulfate</td>
			<td>Sigma</td>
			<td>D8906</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmon Sperm DNA</td>
			<td>Life Technologies&#160;</td>
			<td>15632-011</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A8531</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl (N)</td>
			<td>Sigma</td>
			<td>38283</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Alcohol</td>
			<td>Sigma</td>
			<td>459844</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma</td>
			<td>322415</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Life Technologies&#160;</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase A</td>
			<td>Sigma</td>
			<td>R4642</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepsin</td>
			<td>Sigma</td>
			<td>P6887</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst-33342</td>
			<td>Thermo Scientific</td>
			<td>62249</td>
			<td>Dissolve Hoechst-33342 in PBS to final concentration of 1 mg/mL.</td>
		</tr>
		<tr>
			<td>Rubber Cement/Cytobond Sealant&#160;</td>
			<td></td>
			<td>2020-00-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Seven Coplin Jars</td>
			<td>Thermo Scientific</td>
			<td>107</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; Dark Humidified chamber</td>
			<td>Sigma</td>
			<td>CLS2551</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slides&#160;</td>
			<td>VWR</td>
			<td>48366067</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry Digital Heat Block</td>
			<td>VWR</td>
			<td>13259</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Fluorescence Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20X Saline-Sodium Citrate (20X SSC)</td>
			<td></td>
			<td></td>
			<td>Combine 175 g of NaCl, and 88.2 g of trisodium citrate with 800 mL of molecular grade H2O. Stir while adjusting to pH 7. Once the all salts dissolve, adjust the volume to 1 L and filter through 0.22 &#181;m Filter paper. Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>&#160;1mg/mL of Rnase A</td>
			<td></td>
			<td></td>
			<td>Dilute 20X SSC buffer to 2X SSC buffer. Dissolve RNase A in 2X SSC buffer to a final concentration of 1 mg/mL. Make fresh solution every time.</td>
		</tr>
		<tr>
			<td>1% Pepsin</td>
			<td></td>
			<td></td>
			<td>Dissolve&#160; pepsin (W/V) in 10 mM HCl to a final concentration of 1 % solution. Make fresh every time.</td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde (4% PFA)</td>
			<td></td>
			<td></td>
			<td>Weigh 4.0 g of PFA in fume hood, add 50 mL of 1xPBS, heat to 60 &#176;C while stirring and adjust the pH with drops of NaOH until all PFA dissolves and the solution becomes clear. Adjust the volume to 100 mL, filter through 0.22 &#181;m Filter paper and store 5 mL aliquots at -20 &#176;C. Thaw aliquot of PFA at 37 &#176;C for 10-15 min for subsequent uses.&#160; Adjust filter size depending on amount of paraformaldehyde.</td>
		</tr>
		<tr>
			<td>Wash Buffer</td>
			<td></td>
			<td></td>
			<td>Combine 100 mL of Formamide (20%) with 2.5 mL of 20X SSC, adjust to pH 7 with 1.0 M HCl and bring the volume to 500 mL with molecular grade H2O.</td>
		</tr>
		<tr>
			<td>Hybridization Buffer</td>
			<td></td>
			<td></td>
			<td>Combine 50% formamide, 10% dextran sulfate, 0.1% SDS, and 300 ng/mL Salmon Sperm DNA in 2X SSC buffer.&#160; Amount of Formamide determine how focused chromatids are, titrate the amount&#160;</td>
		</tr>
		<tr>
			<td>Detection Buffer</td>
			<td></td>
			<td></td>
			<td>Dilute 20X SSC buffer to 4X and add 0.2% Tween-20 to make detection buffer.</td>
		</tr>
		<tr>
			<td>Blocking Buffer</td>
			<td></td>
			<td></td>
			<td>Combine 5% bovine serum albumin (BSA) with 0.2% Tween-20 in 4X SSC buffer.</td>
		</tr>
		<tr>
			<td>Dehydrating Solution</td>
			<td></td>
			<td></td>
			<td>Make 70%, 80%, and 95% ethanol solutions.</td>
		</tr>
	</tbody>
</table>

analysis of line-1 retrotransposition at the single nucleus level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

Um diagrama esquemático do vector de retrotransposição L1 é apresentado na Figura 1. O vector consiste em um gene da neomicina em orientação anti-sentido para L1 ORFs que é interrompida por um intrão de globina em orientação com sentido e colada por DP Sites e SA. Quando integrado de forma estável no cromossoma, L1 ARNm é transcrito a partir do promotor de CMV e combinado L1-5&#39;UTR (Figura 1). Durante o processam...

Watch this Scientific Journal Video about Analysis of LINE-1 Retrotransposition at the Single Nucleus Level at JoVE.com

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Análise de LINE-1 retrotransposição no Núcleo de nível único

Aqui nós utilizamos a metodologia FISH para rastrear LINE-1 retrotransposição no nível núcleos único dos spreads de cromossomos de linhas celulares HepG2 expressam de forma estável LINE-1 sintético.

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

analysis-of-line-1-retrotransposition-at-the-single-nucleus-level

Research

JoVE Journal

Biology

8.3K visualizações.  University of Arizona College of Medicine.  Aqui nós utilizamos a metodologia FISH para rastrear LINE-1 retrotransposição no nível núcleos único dos spreads de cromossomos de linhas celulares HepG2 expressam de forma estável LINE-1 sintético. 

Vídeo: Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

Método para Medição da Cinética de Fusão Viral no Nível única partícula

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

Biologia

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

Análise direta de células individuais por Espectrometria de Massa à pressão atmosférica

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

Estudar proteólise de ciclina B no nível da célula individual em populações de células inteiras

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Agroinfiltration eficiente de plantas para alto nível de expressão transiente de proteínas recombinantes

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Análise da Global RNA Síntese no nível da célula única seguinte hipóxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

Protocolo CUBIC Visualiza Protein Expression na resolução única célula em preparações e peles, inteiros Mount

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Análise da função de células Beta utilizando imagens de cálcio de célula única resolução em ilhotas de Zebrafish

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified &#955; DNA at the Single Molecule Level

The fluorescence microscopy&#160;has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. In single molecule assays for studying DNA-protein interactions, there are two important factors for consideration: the DNA substrate with enough length for easy observation and labeling a protein with a suitable fluorescent probe. 48.5 kb &#955; DNA is a good candidate for the DNA substrate. Quantum dots (Qdots), as a class of fluorescent probes, allow long-time observation (minutes to hours) and high-quality image acquisition. In this paper, we present a protocol to study DNA-protein interactions at the single-molecule level, which includes preparing a site-specifically modified &#955; DNA and labeling a target protein with streptavidin-coated Qdots. For a proof of concept, we choose ORC (origin recognition complex) in budding yeast as a protein of interest and visualize its interaction with an ARS (autonomously replicating sequence) using TIRFM. Compared with other fluorescent probes, Qdots have obvious advantages in single molecule studies due to its high stability against photobleaching, but it should be noted that this property limits its application in quantitative assays.

Here, we present a protocol to study DNA-protein interactions by total internal reflection fluorescence microscopy (TIRFM) using a site-specifically modified &#955; DNA substrate and a Quantum-dot labeled protein.

Visualizando a interação entre a proteína Qdot-etiquetadas e λ Site-specifically modificada de DNA a nível da molécula

The fluorescence microscopy&#160;has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

Sondando cinética mRNA no espaço e no tempo em Escherichia coli usando fluorescência de molécula única de duas cores na hibridização situ

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.