smDNA from pBAD EmaAgo E. coli MG1655 infected with phage P1 second rep
The librariy was prepared according to the previously described bridged (splinted) ligation protocol (Kuzmenko,A. et al., (2020) DNA targeting and interference by a bacterial Argonaute nuclease. Nature, doi: 10.1038/s41586-020-2605-1). For the purpose of visualization, a 5 l aliquot of the smDNA sample (one-quarter of the total volume) was dephosphorylated and radiolabelled as described above. Unlabelled DNA was mixed with the radiolabelled aliquot and an equal volume of sample buffer (8 M urea, 20 mM EDTA, 0.005% bromophenol blue) and resolved under denaturing conditions in a 15% polyacrylamide-urea gel. Gel slices corresponding to 1425-nucleotide smDNAs were crushed and DNA was eluted in 0.4 M NaCl solution overnight at 21 C with constant agitation. After ethanol precipitation, DNA was resuspended in 20 l of milliQ-grade water. Illumina-compatible adaptors were ligated to both ends of smDNA. For this purpose, 8 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 20 l of the purified smDNA solution. The reaction mixture was incubated overnight at room temperature. Ligated DNA fragments were recovered from 10% denaturing PAGE. The libraries were amplified and indexed according to the standard protocol (small RNA sequencing kit, NEB), except that PCR conditions were adjusted in order to prevent library overamplification. A series of test PCR reactions were performed to optimize the amount of adaptor-ligated smDNA to achieve the desired amplification level in 3-4 cycles. smDNA libraries were sequenced using the HiSeq2500 platform (Illumina) in the rapid run mode (50-nucleotide single-end reads).