smDNA from pBAD CmiAgo E. coli BL21
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 34 cycles. smDNA libraries were sequenced using the HiSeq2500 platform (Illumina) in the rapid run mode (50-nucleotide single-end reads).
BL21_pBAD_CmiAgo_1