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[DESCRIPTION]
<p>Staining hundreds to thousands of RNA species each of which requires tens of unique encoding probes requires a very large number of unique oligonucleotide sequences. The sheer number of unique sequences makes traditional solid phase oligonucleotide synthesis prohibitively expensive in most cases. For example, assuming a modest cost of $0.10/base, an encoding probe length of ~100 nt, and the need for ~50,000 unique oligos for a single MERFISH measurement, the cost of the needed oligos would be $500,000! To circumvent this astronomical cost, we have developed a high-throughput approach to generating these probes which utilizes array-derived synthesis of complex oligonucleotide pools. These arrayderived complex oligonucleotide pools that contain ~100,000 custom designed sequences can be purchased for only a few thousand dollars. The challenge to using these pools is that each individual sequence is provided in quantities far too small to be used directly for labeling. Thus, we developed an enzymatic amplification protocol to generate the encoding probes in high quantity sufficient for RNA FISH experiments using the array-derived complex oligonucleotide pools as templates.</p>

[GUIDELINES]
<p style="text-align: justify;">The basic protocol involves four steps, which are described in detail in the following sections. First, the oligopool is amplified via PCR to create template molecules for in vitro transcription. Second, an in vitro transcription produces RNA from these template molecules as well as another ~100-fold amplification. Third, a reverse transcription reaction generates single-stranded DNA from this RNA. Fourth, the RNA template is removed via alkaline hydrolysis. We utilize an RNA intermediate for two reasons: i) in vitro transcription can produce very large quantities of nucleic acid in very small volumes, reducing the amount of material that must be used, and ii) the alkaline susceptibility of RNA allows it to be easily removed from the final DNA probes. Because this reaction involves the use of an RNA intermediate, we recommend using the higher standards of laboratory cleanliness often required for handling RNA. Specifically, all surfaces and pipettes should be cleaned daily using RNase removal solutions and separate stocks of all buffers should be kept solely for RNA work.</p>
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<p><strong>Equipment: </strong></p>
<p> </p>
<p>1. Table top centrifuge</p>
<p>2. qPCR machine or thermocycler</p>
<p>3. 37 °C incubator or water bath</p>
<p>4. 50 °C water bath</p>
<p>5. 95 °C water bath</p>
<p>6. Vacuum manifold (optional)</p>
<p>7. Gel electrophoresis equipment for poly-acrylamide gels (optional)</p>
<p>8. Vacuum concentrator (optional)</p>
<p> </p>
<p> </p>
<p><strong>The protocol workflow is as follows: </strong></p>
<p> </p>
<p style="text-align: justify;"><strong>Amplification of <em>in vitro</em> Template (Steps 1-8) </strong></p>
<p style="text-align: justify;">The first step in this protocol is to use PCR to amplify template molecules for the in vitro transcription. We recommend running this reaction as a limited-cycle PCR, i.e. monitor the status of the reaction in real time with a qPCR machine and remove the samples immediately before the final amplification plateau. We recommend limiting the number of PCR cycles because we have found that over amplification of complex libraries can produce molecules that miss-prime on other molecules, forming long concatemers that both reduce the yield of proper encoding probes and which could produce spurious signals.</p>
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<p><strong><em>In vitro</em> Transcription (Steps 9-14) </strong></p>
<p>The second step of this protocol is a high yield in vitro transcription reaction that further amplifies the template molecules created in Section <strong>Amplification of <em>in vitro</em> Template</strong> as well as converts them into RNA.</p>
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<p style="text-align: justify;"><strong>Reverse Transcription and Purification of Encoding Probes (Steps 15-25) </strong></p>
<p style="text-align: justify;">In this step of the protocol, the large quantities of RNA produced by the high yield in vitro transcription are converted to single-stranded DNA using a reverse transcription reaction. This RNA template is then removed via alkaline hydrolysis, and the final encoding probes are purified and concentrated.</p>

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