title: RNA-seq of ticks: adult male day 7
design: The tick pools were macerated in 600L PBS, and 200L of the supernatant was filtered with a 22m filter. Subsequently, DNA was removed from the samples using Promega's RQ1 RNase-Free DNase reagent according to the manufacturer's instructions. Extraction was carried out with GeneJET RNA Purification Kit following the manufacturer's instructions. To initiate the sequencing process, 13 pools of tick RNA were generated taking into account genus, species, stage and geographic location. RNA concentration and Integrity Number (RIN) was measured by fluorometry using the Qubit with RNA Quantification broad range (BR) and RNA IQ Assay Kit (Thermo Fisher Scientific), respectively. For the preparation of libraries, an input of 250 to 500 ng of total RNA was determined. The samples were processed with the MGIEasy Fast RNA library preparation set under the high-throughput DNA nanoball (DNB) sequencing methodology.Initially, RNA was fragmented into products of approximately 250 nucleotides according to the FCL 150 Paired-End (PE) platform. The first and second strands of DNA were then synthesized with Random Hexamer Primers. The fragments were subjected to a process of end repair (ERAT), ligation of molecular barcodes and amplification of products by PCR. The concentration of the library was obtained with the Qubit dsDNA Quantification Assay Kit. Whereas, the size of the fragments was determined with Fragment Analyzer (Aligent Technologies).According to the concentration and size of the fragments, two pools with a final mass of 1000fmol/pool were obtained for the circularization of DNAs and DNB synthesis (>11ng/l). Finally, the cartridge, flow cell and DNBs were loaded into the MGI-G50 machine to perform NGS of short reads (Shenzhen, China).