TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="font-weight: 400;">This protocol is based upon </span><a href="https://www.nature.com/articles/nmeth.4407?WT.feed_name=subjects_gene-expression" target="_blank"><em><span style="font-weight: 400;">Habib, N et al.,</span></em> </a><em><span style="font-weight: 400;"><a href="https://www.nature.com/articles/nmeth.4407?WT.feed_name=subjects_gene-expression" target="_blank">Nature Methods 14: 955-958, 2017</a> </span></em><span style="font-weight: 400;"> and <a href="https://www.nature.com/protocolexchange/protocols/6163" target="_blank">Basu et al., Protocol Exchange 2017: DroNc-seq step-by-step</a></span><span style="font-weight: 400;"> with adaptions for adult human kidney including:</span> <span style="font-weight: 400;">Tissue mincing, homogenization strokes, addition of protease inhibitor and RNasin and adjustments to strainer size and sequence.</span></p>
<p> </p>

[BEFORE_START]
<p><strong>Buffers and Solutions</strong></p>
<p> </p>
<p><span style="font-weight: 400;">Nuclei lysis buffer 0 (NLB0) working solution</span></p>
<ul>
<li style="font-weight: 400;"><span style="font-weight: 400;">10 ml of Nuclei EZ Lysis Buffer</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">1 tablet of cOmplete ULTRA tablets</span></li>
</ul>
<p> </p>
<p><span style="font-weight: 400;">Nuclei lysis buffer 1 (NLB1) working solution - make 4 ml per &lt; 8 mm</span><sup><span style="font-weight: 400;">3</span></sup><span style="font-weight: 400;"> tissue</span></p>
<ul>
<li style="font-weight: 400;"><span style="font-weight: 400;">4 ml of NLB0</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">20 µl of RNasin Plus</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">20 µl of SUPERaseIN</span></li>
</ul>
<p> </p>
<p><span style="font-weight: 400;">Nuclei lysis buffer 2 (NLB2) working solution - make 4 ml per &lt; 8 mm</span><sup><span style="font-weight: 400;">3</span></sup><span style="font-weight: 400;"> tissue</span></p>
<ul>
<li style="font-weight: 400;"><span style="font-weight: 400;">4 ml of Nuclei EZ Lysis Buffer</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">4 µl of RNasin Plus</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">4 µl of SUPERaseIN</span></li>
</ul>
<p> </p>
<p><span style="font-weight: 400;">Nuclei suspension buffer (NSB)</span></p>
<ul>
<li style="font-weight: 400;"><span style="font-weight: 400;">2 ml of DPBS</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">2 µl of RNasin Plus</span></li>
</ul>

[GUIDELINES]
<p><span style="font-weight: 400;">All steps are performed on ice or in cold room (4ºC) to minimize RNA degradation.</span></p>
<p> </p>
<p><strong>Storage temperatures </strong></p>
<p> </p>
<table style="height: 210px; width: 497px;">
<tbody>
<tr>
<td style="width: 409px;"><strong>Reagent</strong></td>
<td style="width: 84px;"><strong>Storage</strong></td>
</tr>
<tr>
<td style="width: 409px;">1x DPBS</td>
<td style="width: 84px;">4<span style="font-weight: 400;">°</span><span style="font-weight: 400;">C</span></td>
</tr>
<tr>
<td style="width: 409px;"><span style="font-weight: 400;">Nuclei EZ Lysis Buffer</span></td>
<td style="width: 84px;"><span style="font-weight: 400;"><span style="font-weight: 400;">4</span></span><span style="font-weight: 400;">°C</span></td>
</tr>
<tr>
<td style="width: 409px;"><span style="font-weight: 400;">cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack</span></td>
<td style="width: 84px;"><span style="font-weight: 400;"><span style="font-weight: 400;">4</span></span><span style="font-weight: 400;">°C</span></td>
</tr>
<tr>
<td style="width: 409px;"><span style="font-weight: 400;">RNasin Plus Ribonuclease Inhibitors</span></td>
<td style="width: 84px;"><span style="font-weight: 400;">-20</span><span style="font-weight: 400;">°</span><span style="font-weight: 400;">C</span></td>
</tr>
<tr>
<td style="width: 409px;"><span style="font-weight: 400;">SUPERaseIN RNase Inhibitor</span></td>
<td style="width: 84px;"><span style="font-weight: 400;">-20</span><span style="font-weight: 400;">°</span><span style="font-weight: 400;">C</span></td>
</tr>
<tr>
<td style="width: 409px;"><span style="font-weight: 400;">RNase free H</span><sub><span style="font-weight: 400;">2</span></sub><span style="font-weight: 400;">O</span></td>
<td style="width: 84px;">RT</td>
</tr>
<tr>
<td style="width: 409px;"><span style="font-weight: 400;">RNaseZap</span></td>
<td style="width: 84px;">RT</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>

[STEPS]
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