title: RNA-seq of Xylella fastidiosa OMVs and WCL
design: RNA-seq of Xylella fastidiosa OMVs and WCL
library_construction_protocol: For WCL samples, 2 mL cultures were centrifuged for 15 min, 4'000 x g. Supernatants were removed and pellets were flash-frozen and kept at -80 °C until further use. For Xf-OMV isolation, the remaining of 200 mL cultures were centrifuged at 4'000 x g for 15 min, supernatants were filtered through 0.22 µm filters (Milipore, SEGTPT0045). Filtered supernatants were centrifuged at 38'000 x g for 1 h to remove cellular debris and then further centrifuged at 150'000 x g for 4 h. Then, pellets were resuspended in 2 mL filtered 1x PBS (pH 7.4) and further purified using qEV2 iZON SEC-columns (iZon qEV2 columns, 70 nm series, IC2-70, France). Xff-OMVs were isolated from 2 x 200 mL cultures and concentrated using iZon magnetic concentrator beads. Xylella fastidiosa fastidiosa subsp. Temecula 1 was routinely grown for 5-10 days on Pierce's Disease 3 (PD3) plates (Davis, 1980). For liquid cultures, 200 mL PD2 (Davis, 1980) was inoculated with resuspended Xf inoculum in PBS and cultures were grown for ca. 4-7 days in PD2 (+ 50 µg/mL for Temecula 1-GFP) at 28 °C and 140 rpm reaching an OD600 >  0.2.  For RNA isolation, concentrated Xff-OMVs were directly resuspended in 1 mL Trizol and kept at 4 °C until further processing. RNA was isolated using Trizol procedure and RNA was purified using RNA Clean & Concentrator Kit with DNAse treatment (ZymoResearch, R1013). Total RNA was isolated from WCL and OMV samples. Concentrated Xff-OMVs were directly resuspended in 1 mL Trizol and kept at 4 °C until further processing. RNA was isolated using Trizol procedure and RNA was purified using RNA Clean & Concentrator Kit with DNAse treatment (ZymoResearch, R1013). The RNA integrity was confirmed using a Bioanalyzer (Agilent). For total RNA-seq of WCL samples, rRNA was depleted using Illumina Ribo-Zero Plus rRNA Depletion Kit (Illumina, #20040526). All cDNA libraries were prepared using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB; #E7760).