DNA 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. The sample was treated with RNase A (ThermoFisher) and precipitated with ethanol. Illumina-compatible adaptors were ligated to both ends of smDNA. For this purpose, 4 l of 5 Rapid Ligation buffer (ThermoFisher), 2 l of 100 M 5-adaptor, 2 l of 100 M of 3-linker, 2 l of 100 M oligonucleotide bridge 1, 2 l of 100 M oligonucleotide bridge 2 and 2 units of T4 PNK (ThermoFisher) were added to 10 l of the purified smDNA solution. The reaction mixture was incubated overnight at room temperature. Ligated DNA fragments were resolved in 10% denaturing PAGE. Gel slices containing ligated DNA were crushed and DNA was eluted in 0.4 M NaCl solution overnight at 21C with constant agitation. After ethanol precipitation, DNA was resuspended in 20 l of nuclease-free water. The libraries were amplified and indexed according to the standard protocol (TruSeq small RNA sequencing kit, NEB), except that PCR conditions were adjusted in order to prevent library overamplification. Adaptor-ligated DNA was cleaned from fragments shorter than 110 bp with NEB Next sample purification beads.