RNA Imaging with MERFISH - Design of Oligonucleotide Probes v1 (protocols.io.menc3de)
The first step in any MERFISH experiment is the design of the oligonucleotide probes that will be used to label individual RNA species. In our current implementation of MERFISH, each oligonucleotide encoding probe consists of three basic components as illustrated in Figure 2 . The first region is a 30-nt targeting region that is complementary to a portion of the sequence of the RNA to which it is designed to bind. The second region is a set of sequences that are called readout sequences, which
... t sequences, which were designed to be complementary and hence only bind to MERFISH readout probes and not other nucleic acid in the cell. Finally, the third region is a set of priming regions used in the construction of these probes, which will be discussed in detail in Probe Construction. In addition to the nucleotide sequences for each of these components, a codebook -the specific set of binary barcodes that will be used and their association with different RNA species of interest-must also be designed. In this section, we provide protocols to design these sequences and to build a codebook. Example code to perform these steps can be found at http://zhuang.harvard.edu/merfish/ . Je ffre y R. Moffi tt, Xi a owe i Zhua ng. Je ffre y R. Moffi tt, Xi a owe i Zhua ng. De si gn of T a rge t Re gi ons De si gn of T a rge t Re gi ons Functionally, the goal of a target region is to direct the binding of each encoding probe to its target RNA of interest with high binding efficiency and specificity. The central challenge in the design of target regions for MERFISH (and smFISH, in general), is to design a set of target regions where these properties are optimized for all probes under a constant set of hybridization conditions, e.g. incubation temperature. To accomplish this goal target regions are designed to cover a relatively narrow range of GC content and melting temperatures (TM ) with their target. In addition, a good target region should have limited homology to other RNAs in the transcriptome, reducing the probability that it will bind to the wrong RNA. Finally, the typical smFISH measurement does not bind each RNA with a single probe but rather tiles that RNA with multiple probes, each of which targets a different portion of the sequence of the RNA. See Figure 2 . For many smFISH measurements, the number of unique probes per RNA is often ~50; however, this number can be lowered with a corresponding reduction in the brightness of the individual RNA spots (Raj et al., 2008). For initial MERFISH work, we recommend having at least 50 encoding probes per RNA. 1 1 03/29/2018 This is an open access protocol distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Asse mbl y a nd Scre e ni ng of Encodi ng Probe s Asse mbl y a nd Scre e ni ng of Encodi ng Probe s Once the target regions, the readout regions, and the barcodes associated with the desired encoding scheme are designed, the sequence of the encoding probes can be assembled. Each encoding probe will contain multiple readout sequences. However, given length restrictions in synthesis of these sequences, it is typically not the case that all of the readout sequences for each RNA will fit into each encoding probe. We use two readout sequences per encoding probe. De si gn of Pri mi ng Re gi ons De si gn of Pri mi ng Re gi ons The protocol that we use to make encoding probes that contain the sequences designed above require the addition of two priming regions to each probe. The optimal regions should have similar TM , no contiguous stretches of the same nucleotide longer than three, and relatively narrow GC content. They should also have limited homology to each other and to non-priming regions of the encoding probes. 1 Download and install all necessary software. OligoArray2.0 can be downloaded from: http://berry.engin.umich.edu/oligoarray2_1/ . This software requires OligoArrayAux which can be downloaded from: http://unafold.rna.albany.edu/q=DINAMelt/OligoArrayAux. OligoArray2.0 will look for this software in a specific directory (C:\Program Files \OligoArrayAux\), so it must be installed there. Finally, OligoArray2.0 also requires several legacy BLAST functions, which can be downloaded and installed from: http:// blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastDocs&DOC_TYPE=Download .