Trancriptome of purified EBs and RBs of C. psittaci 02DC15

The study aimed to elucidate the transcriptome of infectious and non-infectious particles from zoonotic C. psittaci 02DC15, C. abortus S26/3, and 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.