Amplicon sequencing (Positive control, PCR products of synthetic DNA instead of DNA from injected cells)
A 30-bp IES (IESPGM.PTET51.1.160.128593 in Pramecium Databse) along 10bp upstream and downstream flaning sequence was synthesised (Twist Bioscience) with two 30bp of flanking MAC sequence from other chromatin on each side. Four ApT sites within the IES were chemically modified to contain N-methyladenine on both strands (custom synthesis, Genscript). A 3-bp barcode distinguished methylated and unmethylated versions. Equal amounts were mixed and injected into the cytoplasm of Dcl5-silenced cells at mid-late autogamy stage (15-20h after starvation) as described3. Injected cells were allowed to complete development and were grown for ~10 vegetative divisions before DNA extraction.