Readout Probe Design for seqFISH+

Readout probes of 12–15-nt in length were designed for seqFISH as previously described^{20 (link),21 (link)}. In brief, a set of probe sequences was randomly generated with combinations of A, T, G or C nucleotides with a GC-content range of 40–60%. To minimize cross-hybridization between the readout probes, any probes with ten or more contiguously matching sequences between the readout probes were removed. The readout probes for sequential immunofluorescence were similarly designed except ‘C’ nucleotide is omitted^{60 (link)}. The 5’ amine-modified DNA oligonucleotides (Integrated DNA Technologies) with the readout probe sequences were conjugated in-house to Alexa Fluor 647-NHS ester (Invitrogen A20006) or Cy3B-NHS ester (GE Healthcare PA63101) or Alexa Fluor 488-NHS (Invitrogen A20000) as described before^{20 (link),21 (link)}, or fluorophore conjugated DNA oligonucleotides were purchased from Integrated DNA Technologies. In total, 240 unique readout probes^{21 (link)} were designed and synthesized for DNA seqFISH+ experiments, and subsets of those readout probes were used for RNA seqFISH experiments. The cost for 240 readout probes for DNA seqFISH+ were approximately $15,000 with 5’ amine-modified DNA oligonucleotides and dye conjugation in-house, and $50,000 with fully labeled purchase, which can be used over hundreds or thousands of experiments.