Qubit fluorometer 2

Microalgal cells were collected from liquid BG11 medium by centrifugation at 300 rpm at room temperature. Genomic DNA was then extracted using a modified cetyltrimethylammonium bromide (CTAB) method^{32 (link)}. The concentration of extracted DNA was determined by Qubit Fluorometer 2.0 (Life Technologies) and the quality was determined by electrophoresis analysis. A whole-genome shotgun strategy was applied to the qualified samples with large DNA fragments and adequate quantity, including pair-ended libraries with insert sizes ranging between 350 and 500 bp and mate-paired libraries with insert sizes of 2 kb, 5 kb, 10 kb and 20 kb, respectively. Whole genome sequencing was performed on the Illumina sequencers (HiSeq 2500 and HiSeq 4000) at BGI-Shenzhen.
To ensure high-quality of DNA sequencing reads (also called clean data) for downstream analyses, a strict filtering process was carried out using SOAPfilter (V2.2). Briefly, a series of filtering steps were performed on the raw reads, which include removal of Ns-rich (if Ns > 10%) reads, low quality reads (if >10% of the bases for each read are defined as low-quality), reads with ≥10 bp aligned to the adapter sequences and PCR duplicates. Low quality read ends were also trimmed off by 5–8 bp.

The amplicon libraries were prepared using Nextera XT Index Kit (Illumina Inc., San Diego, California, USA) as per the ITS Metagenomic Sequencing Library preparation protocol (Part # 15044223 Rev. B). The amplicons with the Illumina adaptors were amplified using i5 and i7 primers that add multiplexing index sequences as well as common adapters required for cluster generation (P5 and P7). The amplicon libraries were purified by 1X AMpureXP beads, checked on Agilent DNA 1000 chip on Bioanalyzer 2100 and quantified by Qubit Fluorometer 2.0 using Qubit dsDNA HS Assay kit (Life Technologies, India). After obtaining the Qubit concentration for the library and the mean peak size from Bioanalyser profile, the library was spiked with 50% PhiX control v3 (FC-110-3001) as described in the Illumina procedure and loaded onto illumina NGS platform at an appropriate concentration (10–20 pM) for cluster generation and sequencing. The libraries were sequenced at Xcelris Genomics Pvt. Ltd. (Ahmedabad, India), using Illumina HiSeq 2 X 250 base pair chemistry. Sequencing of PCR negative reactions did not yield any fungal reads.

The primers 460F (5′-CCTACGGGNBGCASCAG-3′) and 460R (5′-GACTACNVGGGTATCTAATCC-3′) were used to amplify the V3-V4 hyper-variable region of the 16S rDNA gene of bacteria and archaea [42 (link)]. The amplicons were amplified using i5 and i7 primers as per the standard Illumina protocol [42 (link)]. The amplicon library was prepared with Nextera XT Index Kit (Illumina Inc., Hayward, CA, USA) as per the 16S Metagenomic Sequencing Library preparation protocol with a 2 × 150 read length. The amplicon libraries were purified by 1X AMpureXP beads, checked on Agilent DNA1000 chip on Bioanalyzer2100, and quantified by Qubit Fluorometer 2.0 using Qubit dsDNA HS Assay kit (Life Technologies, Carlsbad, CA, USA).

Multiplex long-range PCR primers (One Lambda, Canoga Park, CA) were designed to co-amplify HLA-A, -B, -C from promoter to 3’-UTR. For HLA class II genes, long-range PCR primers amplified HLA-DRB1 and DQB1 beginning at exon 2 through part of exon 4 (One Lambda, Canoga Park, CA) in separate reactions (Fig 1). In brief, each PCR reaction consisted of: 5μL of buccal swab gDNA, 4μL polymerase buffer, 1.6μL dNTP mixture, 5μL primer mix, and 0.8μL PrimeSTAR GXL DNA polymerase (TaKaRa Bio Inc., Japan), making a final volume of 20μL. GeneAmp PCR system 9700 (Life Technologies, Carlsbad, CA) thermal cycler conditions for class I genes were: 94°C for 2 min, followed by 35 cycles of (98°C for 10 s, 70°C for 3 min). For class II, the optimized conditions were: 94°C for 2 min, followed by 35 cycles of (98°C for 10 s, 69°C for 3 min). PCR products were confirmed on 0.8% agarose gel (Sigma–Aldrich, St. Louis, MO), and then mixed with Agencourt AMPure XP beads (Beckman Coulter, Brea, CA) in 0.6× PCR reaction volume to undergo purification using Biomek NX (Beckman coulter, Brea, CA). Qubit fluorometer 2.0 (Life technologies, Grand Island, NY) was used to quantitate and normalize amplicon concentrations, accounting for the different amplicon fragment lengths; this was followed by equimolar pooling of class I and class II PCR products.

Total RNA was extracted using Qiagen RNeasy kit protocols (Qiagen, Hilden, Germany). The frozen punched samples were allowed to thaw to ambient temperature and QIAzol phase later was separated with 350 µl chloroform (15 min, 12,000 g, 4 °C). The upper aqueous phase was removed then mixed with 70% (v/v) ethanol to precipitate the total RNA, which was resuspended and applied to RNeasy columns in accordance with the manufacturer’s instruction. For RNAseq experiments, punched samples at QIAzol phase were pooled (5 per group), whereas individual samples were used in qRT-PCR (n = 6). The RNA concentration was measured using a Nanodrop spectrophotometer (ND-2000, Thermo Scientific, Wilmington, DE) and Qubit Fluorometer 2.0 (Life Technologies). The RNA samples were also analysed using 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA 95051) to obtain RNA integrity numbers (RIN numbers) as a measure of their quality (Schroeder et al., 2006 (link)). All RNA samples met the RIN quality criterion of >8.5.

Twenty male and females were removed, respectively, at intervals during feeding to recover material representing unfed (day 0 weighing < 5 mg), early feeding (day 2 weighing 6–15 mg for females and < 5 mg for males), mid feeding (day 4 weighing 16–23 mg for females and ~ 5 mg for males) and late feeding (day 6 weighing > 24 mg for females and ~ 7 mg for males) ticks. Salivary glands were dissected out within two hours of tick removal, cleaned from other internal tissues and placed into at least ten times its volume RNAlater (Qiagen, AMBION, Inc., Austin, Texas), and left overnight at 4 °C before storing at -70 °C. Each day’s samples were pooled by gender to render one male (40 glands) and one female (40 glands) sample per time point. Salivary glands were suspended in 600 µl RLT buffer (RNeasy Protect Mini Kit) per 20 mg tissue and total RNA extracted using the RNeasy Protect Mini Kit (QIAGEN Group). Residual genomic DNA was removed with DNase I digestion (QIAGEN Group). Total RNA quantification and assessment of the integrity of RNA was done using the Qubit fluorometer 2.0 (Life Technologies, Carlsbad, CA) and the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA).