Trancriptome of purified EBs and RBs of W. chondrohpila 2032/99

Trancriptome analysis of purified EBs and RBs of W. chondrohpila 2032/99. Therefore EB and RB pellets were incubated in 500 µL TRIsureTM (Bioline) reagent and lysed at 65°C for 5 min followed by phenol chloroform extraction. The aqueous phase was mixed with 1/10 sodium acetate (3M; pH = 5.2) and precipitation was carried out with ethanol. In total 10 µg of RNA per sample was treated with 10 U of DNase I (Thermo Scientific) for 45 min at 37°C. After DNase I digestion RNA molecules longer than 200 nucleotides were purified using RNA Clean & ConcentratorTM-5 kit (ZymoResearch). The absence of DNA was ensured by qPCR. To remove rRNAs the ScriptSeqTM Complete Gold Kit (Epidemiology) was used. RNA quality controls were performed after total RNA isolation, DNA digestion and rRNA removal using Bioanalyzer 2100 measurements and the Agilent RNA 6000 Pico kit (Agilent Technologies). For the synthesis of cDNA libraries, 5 ng chemically fragmented RNA was applied as described in ScriptSeqTM Complete Gold Kit (Illumina). The cDNA libraries were barcoded using ScriptSeqTM Index PCR Primers (Illumina). After PCR amplification cDNA libraries were purified using AMPure XP System (Beckman Coulter). The size distribution of cDNA and absence of primer dimers were monitored via the Agilent High Sensitivity DNA Kit (Agilent Technologies). Final libraries were sequenced by StarSEQ GmbH (Mainz) using a NextSeq 500 (Illumina) platform and the 150 bp paired-end protocol.针对纯化的W. chondrohpila 2032/99菌株的原体（EB，elementary body）与网状体（RB，reticulate body）开展转录组分析。将EB与RB沉淀加入500 μL TRIsure™（Bioline）试剂中孵育，于65℃裂解5分钟后，采用酚-氯仿法进行抽提。取水相并加入1/10体积的3M醋酸钠（pH=5.2），随后以乙醇沉淀RNA。每份样品取总计10 μg RNA，加入10 U脱氧核糖核酸酶I（DNase I，Thermo Scientific），于37℃消化45分钟。DNase I消化完成后，使用RNA Clean & Concentrator™-5试剂盒（ZymoResearch）纯化长度大于200 nt的RNA分子。通过定量PCR（qPCR，quantitative real-time PCR）验证无DNA残留。使用ScriptSeq™ Complete Gold Kit（Epidemiology）去除核糖体RNA（rRNA，ribosomal RNA）。分别在总RNA提取、DNA消化及rRNA去除步骤后，采用Bioanalyzer 2100生物分析仪与Agilent RNA 6000 Pico试剂盒（Agilent Technologies）完成RNA质量控制。参照ScriptSeq™ Complete Gold Kit（Illumina）的操作指南，取5 ng经化学片段化的RNA进行cDNA文库合成。使用ScriptSeq™ Index PCR引物（Illumina）对cDNA文库进行条形码标记。PCR扩增完成后，采用AMPure XP磁珠纯化系统（Beckman Coulter）纯化cDNA文库。采用Agilent High Sensitivity DNA试剂盒（Agilent Technologies）检测cDNA的片段大小分布，并验证无引物二聚体残留。最终文库由位于美因茨的StarSEQ GmbH公司采用Illumina NextSeq 500平台及150 bp双端测序方案完成测序。