Targeted Gene Silencing in Planaria

Two shRNA precursors for each MAC gene were designed as previously described (Karabulut et al. 2019 (link); Lewandowska et al. 2021 (link)). Reverse complement sequence of shRNA precursors were synthesized as DNA ultramer oligo by Integrated DNA Technologies (Coralville, IA), mixed with T7 promoter primer in 1:1 ratio in a final concentration of 25 µM, denatured at 98 °C for 5 min, and cooled to 24 °C. shRNAs were synthesized with AmpliScribe T7-Flash Transcription Kit (Epicentre, Charlotte, NC) for 15 h followed by 15 min treatment with 1 µL of DNase I. The in vitro transcribed products were purified using the Quick-RNA Miniprep Kit (Zymo Research, Irvine, CA). shRNAs were used for microinjection at concentrations ranging from 400 to 1,200 ng/uL. Approximately 100 injected planula (4 dpf) were flash frozen in liquid nitrogen and stored at −80 °C and used for downstream qPCR analysis. MACs1 to 4 were first targeted individually, and then, MAC1, 3, and 4 were targeted simultaneously by combining three validated shRNAs each targeting MAC1, 3, or 4 specifically.