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.

本研究采用Illumina MiSeq测序平台及MiSeq Reagent Kit v3试剂盒，针对高度富集的转运RNA（transfer RNA, tRNA）开展单端RNA测序（Single-end RNA sequencing, 1×100 bp）。针对tRNA文库，采用硼氢化钠（sodium borohydride, NaBH₄）化学处理以检测二氢尿苷、4-硫尿苷及7-甲基鸟苷的tRNA修饰；与未处理的对照样本相比，处理样本中表观读段终止事件的发生频率显著升高，可通过测序比对图谱直观体现上述修饰。针对假尿苷的谱分析，则采用经1-环己基-(2-吗啉代乙基)碳二亚胺对甲苯磺酸盐（1-cyclohexyl-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate, CMCT）处理的tRNA文库完成。
本实验旨在探究亲缘关系密切的芽孢杆菌目（Bacillales）菌株的tRNA依赖型温度适应机制，涵盖三类不同温度偏好的菌株：嗜冷菌（Planococcus halocryophilus与Exiguobacterium sibiricum）、嗜温菌（枯草芽孢杆菌Bacillus subtilis）以及嗜热菌（嗜热脂肪地芽孢杆菌Geobacillus stearothermophilus）。各菌株分别在10℃至70℃的梯度温度下培养，覆盖各自实验室条件下的最适生长温度、最低与最高可生长温度：P. halocryophilus的培养温度为10℃、20℃与30℃；E. sibiricum为10℃、20℃与30℃；枯草芽孢杆菌B. subtilis为20℃、30℃与37℃；嗜热脂肪地芽孢杆菌G. stearothermophilus为40℃、55℃与70℃。
对于每个经NaBH₄处理、CMCT处理的样本，以及对应各菌株在各培养温度下的未处理阴性对照，均设置3次生物学重复；每个物种总计构建36个RNA测序文库。