Expression profiling of Mycobacterium smegmatis in M63 medium and M63 medium supplemented with NH4Cl for wild-type, eccCa1, eccD, esxB. Expression profiling of Mycobacterium smegmatis in M63 medium and M63 medium supplemented with NH4Cl for wild-type, eccCa1, eccD, esxB

Mycobacterium smegmatis respond to various environmental stimuli via ESX systems. We probed the transcriptional response to nitrogen addition to the medium using a series of ESX-1 knockout mutants. Overall design: Expression profiling of Mycobacterium smegmatis in M63 medium and M63 medium supplemented with NH4Cl for wild-type and eccCa1, eccD, esxB mutants RNA was extracted from cultures grown to OD600 nm ~ 0.7-0.8. Three biological replicates (single colonies) were prepared for every strain assayed. The NEB RNA extraction kit was used as directed except for the lysis step, which was completed by bead beating the cells with 0.1 mm zirconia beads. To deplete rRNA, we used the NEB rRNA kit as directed. Samples were prepared using the NEBNext Ultra RNA library prep kit for Illumina, and barcoding oligos were used to pool the libraries. Two technical replicates were prepared for analysis. The resulting pools were sequenced with the Illumina NovaSeq in 75 nt single end reads, resulting in 15M reads per sample.

耻垢分枝杆菌（Mycobacterium smegmatis）可通过ESX系统响应多种环境刺激。本研究利用一系列ESX-1敲除突变体，探究了该菌在培养基中添加氮源后的转录响应特征。

整体实验设计：对野生型及eccCa1、eccD、esxB突变株的耻垢分枝杆菌，分别在M63培养基与添加氯化铵的M63培养基中开展表达谱分析。

将培养至光密度OD600 nm约0.7~0.8的菌体培养物提取RNA。每个待测菌株均设置3次生物学重复（单菌落来源）。严格按照说明书操作，使用NEB RNA提取试剂盒完成RNA提取，仅裂解步骤采用0.1 mm氧化锆微珠对菌体进行珠磨破碎。为去除核糖体RNA（rRNA），我们严格参照说明书使用NEB rRNA去除试剂盒进行处理。采用适配Illumina平台的NEBNext Ultra RNA文库制备试剂盒构建样本文库，并通过条形码寡核苷酸实现文库混合。每个样本设置2次技术重复用于后续分析。最终使用Illumina NovaSeq测序平台对混合文库进行75 nt单端读长测序，每个样本可获得约15百万条读段。