RNA copurified with dAmAgo expressed in E. coli MG1655
E. coli MG1655 Z1 strains carrying the expression plasmids with AmAgo and Agap were grown in 1 liter of LB medium with 50 g/ml kanamycin at 37C until OD600 0.4, cooled down to 30C, induced by the addition of L-rhamnose to 0.1% and grown for 4 h at 30C. The cells were collected by centrifugation at 7,000g, 4C for 15 min, resuspended in lysis buffer (50 mM Tris-HCl, 250 mM NaCl, 5% glycerol, pH 7.4) and lysed in Cell Disruptor CF (Constant Systems) at 30 kpsi. Purification of AmAgo by Ni2+-affinity chromatography was performed as described in Kuzmenko et al., 2020. To prepare RNA sequencing library the first half was treated with DNase I (ThermoFisher), followed by Terminator treatment, phosphorylation, adaptor ligation and cDNA synthesis as described above.