unfed female Ixodes ricinus tick previously reared in vitro without antibiotics (IVG-)
Genomic DNA extraction was performed by the Nucleospin Tissue XS kit (Macherey-Nagel) after washing tick samples in 1% bleach solution and tick tissue preparation. After quality control, DNA was diluted to 1 ng/ L. To amplicon sequencing of the V3-V4 region of bacterial 16S rRNA was performed by primers 341F (5-CCTAYGGGRBGCASCAG-3) and 806R (5-GGACTACNNGGGTATCTAAT-3). The PCR was performed using Phusion High-Fidelity PCR Master Mix (New England Biolabs) followed by agarose gel electrophoresis. PCR product mixtures were purified using the Qiagen Gel Extraction kit (Qiagen, Germany). Libraries were genereted by NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs) and quantified by Qubit and qPCR. Sequencing depth was set as 50k raw reads per sample.