title: Amplicon sequencing (rep. 1)
design: 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). Injected cells were allowed to complete development and were grown for ~10 vegetative divisions before DNA extraction. The PCR pruducts quantity, purity, and integrity of the PCR products were assessed using a Qubit 4.0 fluorometer (dsDNA HS/BR Assay Kit; Thermo Fisher Scientific), a DeNovix DS-11 FX spectrophotometer, and the Agilent FEMTO Pulse System (Genomic DNA 165 kb Kit), respectively. Sequencing libraries were generated using the NEBNext Ultra II FS DNA Library Prep Kit for Illumina (NEB, E7805L) with Unique Dual Index UMI Adaptors (NEB, E7395L), following the manufacturers instructions (Version 4.0_7/23), using 50 ng input DNA and 5 PCR indexing cycles. Library quality was evaluated with a Qubit dsDNA HS Assay (Thermo Fisher Scientific) and an Agilent Fragment Analyzer (HS NGS Fragment Kit). Pooled libraries were sequenced in paired-end mode (151:20:8:151) using NextSeq1000 P2 Reagent Kit (300 cycles) on a NextSeq1000 instrument and using MiSeq i100 Series 5M Reagent Kit (300 cycles) on a MiSeq i100 Plus instrument (Illumina). All DNA QC and sequencing were performed at the Next Generation Sequencing Platform, University of Bern, Switzerland.