TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="font-weight: 400;">Single-cell RNA sequencing (scRNA-seq) has emerged as a central genome-wide method to characterize cellular identities and processes. Consequently, improving its sensitivity, flexibility and cost-efficiency can advance many research questions. Among the flexible plate-based methods, “Single-Cell RNA-Barcoding and Sequencing” (SCRB-seq) is one of the most sensitive and efficient ones. Here, we systematically evaluated experimental conditions of this protocol and find that adding polyethylene glycol considerably increases sensitivity by enhancing cDNA synthesis. Furthermore, using Terra polymerase increases efficiency due to a more even cDNA amplification that requires less sequencing of libraries. We combined these and other improvements to a new scRNA-seq library protocol we call “molecular crowding SCRB-seq” (mcSCRB-seq), which we show to be  the most sensitive and one of the most efficient and flexible scRNA-seq methods to date.</span></p>

[BEFORE_START]
<p>Wipe bench surfaces with RNAse Away and keep working environment clean.</p>

[GUIDELINES]
<ul>
<li>For troubleshooting help, feel free to join our <a href="https://publicslack.com/slacks/mcscrb-seq/invites/new" target="_blank" rel="noopener noreferrer">mcSCRB-seq Slack channel</a>, leave your question in the comments section, or message us directly.</li>
</ul>
<p> </p>
<ul>
<li>The complete list of reagents and plastic ware with order numbers can be found in the 'Materials' section.</li>
</ul>
<p> </p>
<ul>
<li>Make sure all steps involving single-cell lysate and RNA before reverse transcription are carried out swiftly.</li>
</ul>
<p> </p>
<ul>
<li>Size selection of libraries is optional, but has in our experience improved reliability in cluster densities when sequencing. </li>
</ul>
<p> </p>
<ul>
<li>All primer sequences are listed below:</li>
</ul>
<p> </p>
<table style="height: 149px; width: 628px;">
<tbody>
<tr style="height: 20px;">
<td style="width: 101px; height: 20px;"><strong>Oligo</strong></td>
<td style="width: 102px; height: 20px;"><strong>Vendor</strong></td>
<td style="width: 102px; height: 20px;"><strong>Purification</strong></td>
<td style="width: 102px; height: 20px;"><strong>Concentration</strong></td>
<td style="width: 301px; height: 20px;"><strong>Sequence</strong></td>
</tr>
<tr style="height: 20px;">
<td style="width: 101px; height: 20px;">
<div title="Page 9">
<div>
<div>
<p>barcoded oligo-dT (E3V6NEXT)</p>
</div>
</div>
</div>
</td>
<td style="width: 102px; height: 20px;">IDT</td>
<td style="width: 102px; height: 20px;">
<div title="Page 9">
<div>
<div>
<p>TruGrade</p>
</div>
</div>
</div>
</td>
<td style="width: 102px; height: 20px;">
<div title="Page 9">
<div>
<div>
<p>2 µM</p>
</div>
</div>
</div>
</td>
<td style="width: 301px; height: 20px;">
<div title="Page 9">
<div>
<div>
<p>Biotin-ACACTCTTTCCCTACACGACGCTCTTCCGATCT[BC6][UMI10][T30]VN</p>
</div>
</div>
</div>
</td>
</tr>
<tr style="height: 20px;">
<td style="width: 101px; height: 20px;">
<div title="Page 9">
<div>
<div>
<p>TSO unblocked (E5V6NEXT)</p>
</div>
</div>
</div>
</td>
<td style="width: 102px; height: 20px;">IDT</td>
<td style="width: 102px; height: 20px;">HPLC</td>
<td style="width: 102px; height: 20px;">100 µM</td>
<td style="width: 301px; height: 20px;">
<div title="Page 9">
<div>
<div>
<p>ACACTCTTTCCCTACACGACGCrGrGrG</p>
</div>
</div>
</div>
</td>
</tr>
<tr style="height: 20px;">
<td style="width: 101px; height: 20px;">PreAmp (SINGV6)</td>
<td style="width: 102px; height: 20px;">IDT</td>
<td style="width: 102px; height: 20px;">Desalted</td>
<td style="width: 102px; height: 20px;">10 µM</td>
<td style="width: 301px; height: 20px;">
<div title="Page 9">
<div>
<div>
<p>Biotin-ACACTCTTTCCCTACACGACGC</p>
</div>
</div>
</div>
</td>
</tr>
<tr style="height: 20px;">
<td style="width: 101px; height: 20px;"> 3' enrichment primer (P5NEXTPT5)</td>
<td style="width: 102px; height: 20px;">IDT</td>
<td style="width: 102px; height: 20px;">HPLC</td>
<td style="width: 102px; height: 20px;">5 µM</td>
<td style="width: 301px; height: 20px;"> 
<div title="Page 9">
<div>
<div>
<p>AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCG*A*T*C*T</p>
</div>
</div>
</div>
</td>
</tr>
<tr style="height: 20px;">
<td style="width: 101px; height: 20px;">
<div title="Page 9">
<div>
<div>
<p>i7 Index Primer (N7XX)</p>
</div>
</div>
</div>
</td>
<td style="width: 102px; height: 20px;">IDT</td>
<td style="width: 102px; height: 20px;">TruGrade</td>
<td style="width: 102px; height: 20px;">5 µM</td>
<td style="width: 301px; height: 20px;">
<div title="Page 9">
<div>
<div>
<p>CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGG</p>
</div>
</div>
</div>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p>Find the cell barcode sequences in the attached text file (Abstract tab).</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.