title: DNA-seq of Xylella fastidiosa OMVs and WCL
design: DNA-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 1x 200 mL cultures and concentrated using iZon 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.  DNA isolation was performed using phenol/chloroform extraction as published in (Spada et al., 2020). Samples were incubated with 100 µL of iZon concentrator beads at RT for 1 h and pelleted at 16'800 x g for 10 min. OMV pellets were resuspended in DNA extraction buffer (SDS 0.5%, Tris-HCL 50 mM pH8, EDTA 0.1 M) and 0.1 mg/mL of ProtK and incubated at 56 °C overnight. Then, exosomal DNA was isolated using phenol/chloroform. DNA was pelleted for 2 h at -80 °C and precipitation was facilitated by addition of 1 µL glycogen (Thermo Scientific, FERR0051). DNA of untreated Xff-OMVs was then used to perform DNA sequencing. Size and concentration of DNA was analyzed using DNA High sensitivity bioanalyzer chip. Sequencing libraries were constructed from 1 ng of DNA with the Nextera XT DNA Sample Preparation Kit (Illumina, Germany) according to the manufacturer's protocol. The library was quality controlled by analysis on an Agilent 2000 Bioanalyzer with the Agilent High Sensitivity DNA Kit (Agilent Technologies, Germany) for fragment sizes of ca. 500-800 bp.