GSM9315536: E. coli, 25%wt+75%ΔmiaA, 42°C, rep2; Escherichia coli; OTHER
Extraction of small RNAs from cells: Small RNAs were extracted according to the manufacturer's protocol (PureLink™ miRNA Isolation Kit (Invitrogen)) with some adjustments. Briefly, cells were suspended in 1 ml Trizol (Ambion). The samples were vortexed for 2 min after adding 0.2 ml chloroform and glass beads, and then were centrifuged at 16 000 rcf, 4°C for 15 min. After adding 215 µl ethanol to the upper phase, the samples were transferred to a spin cartridge and centrifuged at 12 000 rcf for 1 min. 700 µl of ethanol was added to the flow-through, and the samples were transferred to a new spin cartridge followed by centrifugation at 12 000 rcf for 1 min. After washing the cartridge with wash buffer, small RNAs were eluted with 50 µl of DEPC-treated water. For ms2i6A modification detection, in brief, 500 ng of deacylated small RNAs were hybridized with 1 μl of 10 μM tRNATyr stem-loop primer using the following thermocycling program: 95°C for 2 min, 65°C for 30 s, followed by a gradual ramp to 4°C at 5°C per 30 s. After incubation on ice for 1 min, reverse transcription was performed in 20 μl reactions containing 100 μM dA/C/GTP, 12.5 μM dTTP, 100 nM RT-KTq I614Y enzyme, and RT buffer [50 mM Tris-HCl (pH 9.2), 16 mM (NH₄)₂SO₄, 2.5 mM MgCl₂, 0.1% Tween-20], and incubation at 55°C for 1 hour. Following reverse transcription, 5 μl of cDNA was used for PCR amplification in a 50 μl reaction containing 0.5 µM forward and reverse primers, 250 μM dNTPs, 2 mM MgCl2, 1 x Taq buffer with KCl, and 1.25 U Taq polymerase (Thermo Fisher). The thermal cycling conditions were: 96°C for 3 min; 40 cycles of 96°C for 30 s, 50°C for 45 s, and 72°C for 45 s; followed by a final extension at 72°C for 5 min. 3′-end primers included sample-specific barcodes for multiplexing. PCR products were resolved on a 2% agarose gel and purified using the QIAquick Gel Extraction Kit (Qiagen). The purified PCR products were pooled and submitted for Next-generation sequencing. For i6A modification detection, deacylated small RNAs were further treated with 0.5 M I2 for 20 min on ice. Following iodine treatment, saturated Na2S2O3 was added to the reaction mixture until the solution became colorless. Subsequently, saturated Na2CO3 was added dropwise until effervescence ceased. Upon completion of the reaction, small RNAs were purified using the RNA Clean & Concentrator Kit (ZYMO Research) according to the manufacturer's instructions. Iodine-treated samples were then used for sequencing library preparation following the same protocol as described above for ms2i6A detection.