TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Adapted from Core LJ, Waterfall JJ, Lis JT. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science. 2008;322(5909):1845-8. PubMed PMID: 19056941.</p>
<p>This method can be used to detect nascent transcripts. The conditions given are suitable for HeLa cells and should be adapted if other cell types are used. </p>

[BEFORE_START]
<p>Prepare the following reagents in advance and store at +4°C:</p>
<table style="width: 547.703125px;" border="line">
<tbody>
<tr style="height: 88px;">
<td style="width: 220px; height: 88px;">
<p><strong>Swelling buffer: </strong></p>
<p>Tris-HCl, pH 7.5</p>
<p>MgCl<sub>2</sub>  </p>
<p>CaCl<sub>2</sub>     </p>
<p>Water         </p>
</td>
<td style="width: 58px; height: 88px; text-align: right;">
<p><strong>Stock</strong></p>
<p>1 M</p>
<p>1 M</p>
<p>1 M</p>
<p> </p>
</td>
<td style="width: 110px; height: 88px; text-align: right;">
<p><strong>Final Conc </strong></p>
<p>10 mM</p>
<p>2 mM</p>
<p>3 mM</p>
<p>     </p>
</td>
<td style="width: 157.703125px; height: 88px; text-align: right;">
<p><strong>Vol </strong><strong>(for 50 ml)</strong></p>
<p>500 µl</p>
<p>100 µl</p>
<p>150 µl</p>
<p>to 50 ml</p>
</td>
</tr>
<tr style="height: 96px;">
<td style="width: 220px; height: 96px;">
<p><strong>Lysis Buffer: </strong></p>
<p>Swelling buffer  +</p>
<p>Igepal  </p>
<p>Glycerol  </p>
<p>RNasin</p>
<p>Water</p>
</td>
<td style="width: 58px; height: 96px; text-align: right;">
<p><strong>Stock</strong></p>
<p> </p>
<p>50 %</p>
<p>100 %</p>
<p>40 U/µl</p>
<p>   </p>
</td>
<td style="width: 110px; height: 96px; text-align: right;">
<p><strong>Final Conc</strong></p>
<p> </p>
<p>0.5 %</p>
<p>10 %</p>
<p>2 U/ml</p>
<p>                                         </p>
</td>
<td style="width: 157.703125px; height: 96px; text-align: right;">
<p><strong>Vol </strong><strong>(for 50 ml)</strong></p>
<p> </p>
<p>500 µl</p>
<p>5 ml</p>
<p>100 U</p>
<p>to 50 ml</p>
</td>
</tr>
<tr style="height: 100px;">
<td style="width: 220px; height: 100px;">
<p><strong>Freezing Buffer: </strong></p>
<p>Tris-HCl pH 8.3</p>
<p>Glycerol  </p>
<p>MgCl<sub>2</sub></p>
<p>EDTA</p>
<p>Water</p>
</td>
<td style="width: 58px; height: 100px; text-align: right;">
<p><strong>Stock</strong></p>
<p>1 M</p>
<p>100 %</p>
<p>1 M</p>
<p>0.5 M</p>
<p> </p>
</td>
<td style="width: 110px; height: 100px; text-align: right;">
<p><strong>Final Conc</strong></p>
<p>50 mM </p>
<p>40 %</p>
<p>5 mM</p>
<p>0.1 mM</p>
<p> </p>
</td>
<td style="width: 157.703125px; height: 100px; text-align: right;">
<p><strong>Vol </strong><strong>(for 50 ml)</strong></p>
<p>2.5 ml</p>
<p>20 ml</p>
<p>250 µl</p>
<p>10 µl</p>
<p>to 50 ml</p>
</td>
</tr>
<tr style="height: 176px;">
<td style="width: 220px; height: 176px;">
<p><strong>Assay Buffer: </strong></p>
<p>Tris-HCl pH 8.0</p>
<p>MgCl2</p>
<p>DTT</p>
<p>KCl</p>
<p>RNasin</p>
<p>*Sarkosyl                        </p>
<p>ATP, GTP, CTP and UTP/Br-UTP</p>
<p>Water</p>
<p>*Add sarkosyl just before starting incubation</p>
</td>
<td style="width: 58px; height: 176px; text-align: right;">
<p><strong>Stock</strong></p>
<p>1 M</p>
<p>1 M</p>
<p>0.1 M</p>
<p>3 M</p>
<p>40 U/µl</p>
<p>10 %</p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
</td>
<td style="width: 110px; height: 176px; text-align: right;">
<p><strong>Final Conc</strong></p>
<p>10 mM</p>
<p>5 mM</p>
<p>1 mM</p>
<p>300 mM</p>
<p>20 U/ml</p>
<p>1 %</p>
<p>500 µM</p>
<p>       </p>
<p>              </p>
<p>               </p>
</td>
<td style="width: 157.703125px; height: 176px; text-align: right;">
<p><strong>Vol (for 5 ml)</strong></p>
<p>50 µl</p>
<p>25 µl</p>
<p>50 µl</p>
<p>0.5 µl</p>
<p>2.5 µl</p>
<p>500 µl</p>
<p> </p>
<p>to 5 ml</p>
<p><strong> </strong></p>
<p><strong> </strong> </p>
</td>
</tr>
<tr style="height: 96px;">
<td style="width: 220px; height: 96px;">
<p><strong>Binding Buffer for anti BrdU:</strong></p>
<p>SSPE  <strong>  </strong></p>
<p>EDTA</p>
<p>Tween20</p>
<p>NaCl</p>
<p>Water</p>
</td>
<td style="width: 58px; height: 96px; text-align: right;">
<p><strong>Stock</strong></p>
<p>20 x</p>
<p>0.5 M</p>
<p>10 %</p>
<p>5 M</p>
<p> </p>
</td>
<td style="width: 110px; height: 96px; text-align: right;">
<p><strong>Final Conc </strong></p>
<p>0.25 x</p>
<p>1 mM</p>
<p>0.05 %</p>
<p>37.5 mM</p>
<p> </p>
</td>
<td style="width: 157.703125px; height: 96px; text-align: right;">
<p><strong> Vol (for 14 ml)</strong></p>
<p>175 µl</p>
<p>28 µl</p>
<p>70 µl</p>
<p>105 µl</p>
<p>to 14 ml</p>
</td>
</tr>
<tr style="height: 68px;">
<td style="width: 220px; height: 68px;">
<p><strong>Blocking Buffer:</strong></p>
<p>Binding Buffer +</p>
<p>polyvinylpyrrolidone (PVP)</p>
<p>BSA</p>
<p>Water</p>
</td>
<td style="width: 58px; height: 68px; text-align: right;">
<p><strong>Stock</strong></p>
<p> </p>
<p>10 %</p>
<p>50 µg/ml</p>
<p> </p>
</td>
<td style="width: 110px; height: 68px; text-align: right;">
<p><strong>Final Conc</strong></p>
<p> </p>
<p>0.1 %</p>
<p>1 µg/ml</p>
<p> </p>
</td>
<td style="width: 157.703125px; height: 68px; text-align: right;">
<p><strong>Vol (for 2 ml)</strong></p>
<p> </p>
<p>20 µl</p>
<p>40 µl</p>
<p>to 2 ml</p>
</td>
</tr>
<tr style="height: 68px;">
<td style="width: 220px; height: 68px;">
<p><strong>Low Salt Wash Buffer:</strong></p>
<p>SSPE  <strong>  </strong></p>
<p>EDTA</p>
<p>Tween20</p>
<p>Water</p>
</td>
<td style="width: 58px; height: 68px; text-align: right;">
<p><strong>Stock</strong></p>
<p>20 x</p>
<p>0.5 M</p>
<p>10 %</p>
<p> </p>
</td>
<td style="width: 110px; height: 68px; text-align: right;">
<p><strong>Final Conc </strong></p>
<p>0.2 x</p>
<p>1 mM</p>
<p>0.05 %</p>
<p> </p>
</td>
<td style="width: 157.703125px; height: 68px; text-align: right;">
<p><strong>Vol (for 10 ml)</strong></p>
<p>100 µl</p>
<p>20 µl</p>
<p>50 µl</p>
<p>to 10 ml</p>
</td>
</tr>
<tr style="height: 42px;">
<td style="width: 220px; height: 42px;">
<p><strong>High Salt Wash Buffer:</strong></p>
<p>SSPE  <strong>  </strong></p>
<p>EDTA</p>
<p>Tween20</p>
<p>NaCl</p>
<p>Water</p>
</td>
<td style="width: 58px; height: 42px; text-align: right;">
<p><strong>Stock</strong></p>
<p>20 x</p>
<p>0.5 M</p>
<p>10 %</p>
<p>5 M</p>
<p> </p>
</td>
<td style="width: 110px; height: 42px; text-align: right;">
<p><strong>Final Conc </strong></p>
<p>0.25 x</p>
<p>1 mM</p>
<p>0.05 %</p>
<p>100 mM</p>
<p> </p>
</td>
<td style="width: 157.703125px; height: 42px; text-align: right;">
<p><strong>Vol (for 10 ml)</strong></p>
<p>125 µl</p>
<p>20 µl</p>
<p>50 µl</p>
<p>200 µl</p>
<p>to 10 ml</p>
</td>
</tr>
<tr style="height: 1px;">
<td style="width: 220px; height: 1px;">
<p><strong>Elution Buffer :</strong></p>
<p>DTT </p>
<p>NaCl</p>
<p>Tris-HCl pH 7.5</p>
<p>EDTA</p>
<p>SDS</p>
<p>Water</p>
<p> </p>
</td>
<td style="width: 58px; height: 1px; text-align: right;">
<p><strong>Stock</strong></p>
<p>1 M</p>
<p>5 M</p>
<p>1 M</p>
<p>0.5 M</p>
<p>20 %</p>
<p> </p>
<p> </p>
</td>
<td style="width: 110px; height: 1px; text-align: right;">
<p><strong>Final Conc </strong></p>
<p>20 mM</p>
<p>150 mM</p>
<p>50 mM</p>
<p>1 mM</p>
<p>0.1 %</p>
<p> </p>
<p> </p>
</td>
<td style="width: 157.703125px; height: 1px; text-align: right;">
<p><strong>Vol (for 5 ml)</strong></p>
<p>100 µl</p>
<p>150 µl</p>
<p>250 µl</p>
<p>10 µl</p>
<p>25 µl</p>
<p>to 5 ml</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<p>TET:</p>
<p>Tris.EDTA + 0.5 % Tween20</p>
<p> </p>
<p>Add 2 µl of RNasin to all buffers (40 U/µl) unless otherwise stated</p>

[GUIDELINES]
<p>This protocol is for 1 confluent 15 cm<sup>2</sup> dish of HeLa cells. Parameters should be adapted for other cell types.</p>

[STEPS]
?. 
?. 
?. 
?.