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 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 \u2014the specific set of binary barcodes that will be used and their association with different RNA species of interest\u2014must 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\/ .