smiFISH primary probes and FLAPs (secondary probes, either fluorescent or conjugated to digoxigenin) were produced and purchased from Integrated DNA Technologies (IDT), with the following production details. The primary probes are produced using high-throughput oligonucleotides synthesis in 96-well plates. To make use of low-scale synthesis (25 nmol), the total length of primary probes (transcript-binding + FLAP-binding) should not exceed 60 nucleotides for cheaper synthesis. At this scale, oligonucleotides synthesis is possible at the price of ∼0.1 Euros per base at the time of the writing of the paper (∼150 Euros for 24 primary probes). The secondary probes are conjugated to two Cy3, Cy5 or digoxygenin moieties through 5′ and 3′ amino modifications. smFISH probes were synthesized by J.M. Escudier (SPCMIB, Toulouse, France) and labelled with Cy3 mono-reactive dye pack (GE Healthcare).
All primary probes sequences are available online at https://bitbucket.org/muellerflorian/fish_quant in the Oligostan folder. FLAP sequences are listed in Supplementary Note 1.