R8759

The binding buffer included the following: pH 7.4 PBS containing 4.5 mM MgCl₂, 0.005% Tween 20, and 100 μg/mL tRNA (Sigma, R8759). The wash buffer included the following: pH 7.4 PBS containing 4.5 mM MgCl₂ and 0.005% Tween 20. The ssDNA library was as follows: 5′-GGACAGGACCACACCCAGCG-(N40)-GGCTCCTGTGTGTCGCTTTGT-3′; forward primer, 5′-TTTTTTTTTTTTTTTTTTTT/iSp9/ACAAAGCGACACACAGGAGCC-3′; reverse primer, 5′-GGACAGGACCACACCCAGCG-3′. The initial ssDNA library sequence (RNV-75) was synthesized by Integrated DNA Technologies (IDT), with a central 40-nt randomized sequence and primer binding sites on both ends. The reverse primer was conjugated with 20 consecutive dT nt via a triethylene glycol spacer to facilitate denaturing PAGE-based ssDNA separation.⁹ (link)^,33 (link) To prepare biotin-labeled sub-library pools for binding capacity assessment after each round, a forward primer with a 5′-biotin label was synthesized.

Custom Stellaris FISH probes were designed against TAHRE transcripts by using the Stellaris RNA FISH probe designer version 4.1 (Biosearch Technologies, Inc.; available online at http://www.biosearchtech.com/stellarisdesigner). The wild-type, p53⁻, and p53Rescue ovaries were hybridized with the TAHRE Stellaris RNA FISH probe set labeled with Quasar 570 (Biosearch Technologies, Inc.) following the manufacturer's instructions (available online at http://www.biosearchtech.com/stellarisprotocols). Briefly, ovaries were dissected into PBS and fixed for 45 min at room temperature with 4% formaldehyde solution in PBS. After fixation, ovaries were placed in 70% EtOH overnight at 4°C. The following day, the EtOH was aspirated, and wash buffer (2× SSC, 10% deionized formamide in nuclease-free water) was added for 5 min. The probe was diluted at a concentration of 50 nM in hybridization buffer (2× SSC, 10% dextran sulfate [Sigma, D8906], 1 mg/mL tRNA [Sigma, R8759], 2 mM vanadyl robonucleoside complex [New England Biolabs], 10% deionized formamide in nuclease free water). The wash buffer was aspirated, and the hybridization + probe solution was added to each sample and placed for 24 h at 37°C. The samples were then washed with wash buffer twice for 30 min each at 37°C. VectaShield (Vector Laboratories) with DAPI was added before mounting and imaging.