V3-V4 regions of the 16S rRNA gene of Bacteria: Rhipicephalus microplus female
Each tick was washed several times with deionized water before further processing. DNA was extracted using the NucleoSpin tissue kit (Macherey-Nagel GmbH & Co. KG, Duren, Germany) according to the manufacturers instructions. DNA concentration and purity were determined using a NanoDrop One Spectrophotometer (Thermo Fisher Scientific, Waltham, MA). PCR amplification of the V3-V4 regions of the 16S rRNA gene was performed using a pair of region-specific primers 341F (5-CCT AYG GGR BGC ASC AG-3) and 806R (5-GGA CTA CNN GGG TAT CTA AT-3) (NovogeneAIT, Singapore) connected with sample-identifying barcodes. The DNA fragments were end-repaired and A-tailed then further ligated with Illumina adapters. The libraries were generated and sequenced on the Illumina Novaseq 6000 platform (Illumina, USA).