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Most sRNA biogenesis mechanisms involve either RNAse III cleavage or ping-pong amplification by different Piwi proteins harbouring slicer activity. Here, we follow the question why the mechanism of transgene-induced silencing in the ciliate Paramecium needs both Dicer activity and two Ptiwi proteins. This pathway involves primary siRNAs produced from non-translatable transgenes and secondary siRNAs from targeted endogenous loci. Our data does not indicate any signatures from ping-pongdoi:10.6084/m9.figshare.16828960.v1 fatcat:otnufvrf4baxhjhr5mfn735lky