TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Non-enzymatic, high-gain signal amplification methods with single-cell, single-molecule resolution are in great need. We present click-amplifying FISH (clampFISH) for the fluorescent detection of RNA that combines the specificity of oligonucleotides with bioorthogonal click chemistry in order to achieve high specificity and extremely high-gain (&gt;400x) signal amplification. We show that clampFISH signal enables detection with low magnification microscopy and separation of cells by RNA levels via flow cytometry. Additionally, we show that the modular design of clampFISH probes enables multiplexing, that the locking mechanism prevents probe detachment in expansion microscopy, and that clampFISH works in tissue samples.</p>

[BEFORE_START]
<p><strong>Fixing adherent cells:</strong></p>
<p> </p>
<p>Fixation solution (can leave in hood at RT for months):</p>
<ul>
<li>5mL 10x PBS</li>
<li>5mL 37% formaldehyde (100% formalin)</li>
<li>40mL NF H<sub>2</sub>O </li>
</ul>
<p> </p>
<p>70% EtOH (lasts forever):</p>
<ul>
<li>35mL 95% EtOH</li>
<li>15mL Nuclease-free (NF) H<sub>2</sub>O </li>
</ul>
<p> </p>
<p> </p>
<p><strong>Making the probes:</strong></p>
<p> </p>
<p>Materials:<br />1. 5’hexynyl labeled probes with adapter region (left arm)--resuspend in NF H<sub>2</sub>O to 400 μM<br />    a. Could include internal fluor in the left arm if desired (Cy3 or Cy5 from IDT)<br />2. 3’NHS-azide probes with adapter region (right arm)--resuspend in NF H<sub>2</sub>O to 400 μM<br />3. Left and right adapters for each probe--resuspend in NF H<sub>2</sub>O to 400 μM<br />4. Backbone sequence--resuspend in NF H<sub>2</sub>O to 400 μM</p>
<p> </p>
<p> </p>
<p><strong>ClampFISH:</strong></p>
<p> </p>
<p>ClampFISH hybridization buffer (Store at -20°C):</p>
<ul>
<li>10% Dextran sulfate</li>
<li>20% Formamide</li>
<li>2X SSC</li>
<li>NF H<sub>2</sub>O</li>
</ul>
<p> </p>
<p>Wash buffer (Store at RT):</p>
<ul>
<li>10% Formamide</li>
<li>2X SSC</li>
<li>NF H<sub>2</sub>O</li>
</ul>
<p> </p>
<p>Click components:</p>
<ul>
<li>BTTAA (Order through iLab at AECOM Chem bio facility): <a href="http://www.einstein.yu.edu/research/shared-facilities/chemical-biology/Ligands-for-CuAAC/" target="_blank" rel="noopener noreferrer">http://www.einstein.yu.edu/research/shared-facilities/chemical-biology/Ligands-for-CuAAC/</a> Or from Jena bioscience: <a href="https://www.jenabioscience.com/click-chemistry/click-reagents-by-chemistry/auxiliary-cu-i-cli ck-reagents/clk-067-bttaa" target="_blank" rel="noopener noreferrer">https://www.jenabioscience.com/click-chemistry/click-reagents-by-chemistry/auxiliary-cu-i-cli ck-reagents/clk-067-bttaa </a>(Store 20mM aliquot at -20°C)</li>
<li>CuSO<sub>4</sub> (Store 5mM aliquot at -20°C)</li>
<li>Sodium Ascorbate (store at RT)</li>
<li>2X SSC/0.25% Triton (store at RT)</li>
</ul>
<p> </p>
<p> </p>

[GUIDELINES]
<p><strong>The protocol workflow is as follows: </strong></p>
<ol>
<li>Fixing adherent cells (Steps 1-7) </li>
<li>Designing the probes (Raj lab probe design server-- Matlab required) (Steps 8-35)</li>
<li>Making the probes (Steps 36-48) </li>
<li>ClampFISH (Steps 49-90)</li>
</ol>
<p>________________________________________________________</p>
<p> </p>
<p>For additional information and supplementary material, please see full manuscript: </p>
<p><a href="https://www.biorxiv.org/content/early/2017/11/21/222794" target="_blank" rel="noopener noreferrer">https://www.biorxiv.org/content/early/2017/11/21/222794</a></p>

[STEPS]
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