RNA sequencing of tRNA libraries from 4 Bacillales, either untreated or treated with CMCT or NaBH4
Single-end RNA sequencing (1x100 bp) was conducted on highly enriched tRNAs using an Illumina MiSeq with the MiSeq Reagent Kit v3. The tRNA libraries underwent chemical sodium borohydride (NaBH4) treatment to detect dihydrouridine, 4-thiouridine, and 7-methyl-guanosine tRNA modifications in the mapping profile, indicated by an increased occurrence of apparent read terminations compared to untreated control samples. For profiling pseudouridine, sodium borohydride 1-cyclohexyl-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (CMCT) treated tRNA libraries were utilized.The experiment aimed to investigate tRNA-dependent temperature adaptation mechanisms in closely related psychrophilic (Planococcus halocryophilus and Exiguobacterium sibiricum), mesophilic (Bacillus subtilis), and thermophilic (Geobacillus stearothermophilus) Bacillales. Cultures were grown at different temperatures ranging from 10 to 70C, representing optimal growth temperature as well as minimum and maximum feasible temperatures in the laboratory for each species: P. halocryophilus (10C, 20C, and 30C), E. sibiricum (10C, 20C, and 30C), B. subtilis (20C, 30C, and 37C), and G. stearothermophilus (40C, 55C, and 70C).Each NaBH4- and CMCT-treated sample, as well as the corresponding untreated negative control for each bacterium at each cultured temperature, underwent three biological replicates for a total of 36 RNA seq libraries per species.