BioSample

To obtain AfAgo-bound nucleic acids, E. coli BL21(DE3) was transformed with pBAD_TwinStrep-scfAfAgo plasmid. Cells were grown at 37C in LB medium in the presence of 50g/ml ampicillin until OD600=0.7 was reached. Then, expression was induced by adding 0.2% w/v L-arabinose, and cells were harvested after 4h. Cells were disrupted by incubating 1h at 30C in lysis buffer, containing 20mM Tris-HCl (pH8.0 at 25C), 100mM NaCl, 2mM phenylmethylsulphonyl fluoride, 5mM 2-mercaptoethanol 3mg/ml lysozyme (ThermoFisher cat#89833). The scfAfAgo-NA complex was purified using a StrepTactin column, all buffer solutions contained 100mM NaCl. scfAfAgo-bound RNA was purified using phenol-chloroform isolation as described in Zaremba et al., 2021 (doi: https://doi.org/10.1038/s41564-022-01239-0). Then RNA sample was converted to a DNA library using Small RNA-Seq Library Prep Kit (Lexogen cat#052). The resulting DNA library was sequenced using Illumina MiniSeq sequencing with single-end reads and 75bp read length.