RNA from Escherichia coli treated with Agap nuclease rep 2
The Thermo Fisher RNA extraction kit was used to purify total RNA from E. coli BL21(DE3) cells. Next, 5 g of RNA was incubated with either 1 M Agap protein. The reaction was performed in 50 l buffer containing RiboLock RNase inhibitor (Thermo Fisher Scientific) at 37C for 30 minutes. To completely digest all 5-monophosphorylated RNA, 1 U of Terminator 5-phosphate-dependent exonuclease (LGC Biosearch) was added to the mixture, which was then incubated at 37C for 1 hour. The RNA was precipitated with 0.1 volume of 3M sodium acetate and 3 volumes of 96% ethanol in the presence of linear polyacrylamide at -70C. Following centrifugation at 14000 rpm at +4C for 1 hour, the precipitate was dissolved in 10 l of nuclease-free water. Next, the RNA was phosphorylated by T4 PNK enzyme (Thermo Fisher Scientific) in the presence of RiboLock and 1mM ATP. The reaction mixture was incubated at 37C for 30 minutes and subsequently at 65C for 10 minutes. 5'-SR Adaptor for Illumina from NEBNext Small RNA Library Prep Set kit was then ligated to the 5'-end of RNA molecules using T4 RNA ligase 1 (New England Biolabs). The reaction mixture contained 10% DMSO and RiboLock. The ligation reaction mixture was incubated at 25C for 2 hours, followed by a 16-hour incubation at 16C and was stopped by addition of the same volume of stop-solution with urea. The samples were subjected to 10% polyacrylamide gel electrophoresis (PAGE) at 300V for 10 minutes and stained with SybrGold. All gel fragments above the adaptor were excised, and the gel purification was performed by adding 1 ml of 0.4M NaCl and 1 l RiboLock to the gel slice, shaking the mixture overnight at 20C. The purified RNA was precipitated and then resuspended in 11 l of nuclease-free water. cDNA synthesis was conducted using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) following the manufacturer's instructions and employing a primer with a 6N degenerate sequence at its 3'-end. The reaction was terminated by adding an equal volume of stop-solution. Subsequently, the samples were subjected to 10% polyacrylamide gel electrophoresis. Gel fragments located above the primers were excised and extracted overnight at 30C with gentle shaking in 0.4M NaCl. The DNA was precipitated and resuspended in 15 l of nuclease-free water. Finally, PCR amplification was carried out for 15 cycles using Q5 DNA polymerase (New England Biolabs) following the standard protocol and index-containing primers from the NEBNext Small RNA Library Prep Set kit. PCR products were separated on a 6% native gel, and fragments ranging from approximately 135 to 330 nucleotides were excised, precipitated, washed with 80% ethanol, resuspended in 12 l of nuclease-free water, and submitted for single-end Illumina sequencing.