PCR condition is as described in manufacture's instruction except for setting annealing temperatures and PCR cycles as 65°C and 30 cycles.
In Orthodox schools, the Greek language would be the sole medium of instruction, except for grades one through three.
For FF samples, 30-100 ng Ribo-Zero RNA was used for the construction of the library using the Illumina TruSeq™ RNA Sample Prep Kit (Cat# RS-122-2001) and followed the manufacturer's instruction, except for omitting the purification step before fragmentation.
The cDNA libraries were constructed using the UNI-ZAP® XR Vector System (Stratagene, La Jolla, CA) following the manufacturer's instructions, excepting for the libraries L08 and L11, where the kit "Creator Smart cDNA library construction" (Clontech, Mountain View, CA) was used.
The DNA was extracted as per manufacturer's instructions, except for the following modifications: lysis buffer (AP1, RNase A and Reagent DX) and AP2 buffer volumes were increased 2.5× to allow for homogenisation of the tissue (50 mg) with the Geno/Grinder™ 2000 (settings as per Method C).
The DNA was extracted as per manufacturer's instructions, except for the following modifications: volumes of lysis buffers were increased further [773 μL PL2 and 27 μL RNase A (total 800 μL), and 200 μL PL3], but the relative ratios of the individual buffers were maintained as before.
Bisulfite genomic sequencing [20] was performed as described in our previous study [11], or by using an EpiTect Bisulfite Kit (Qiagen) following the supplier's instructions, except for the bisulfite reaction conditions.
5' RACE was performed on 5 µg DNase-treated RNA using the GeneRacer Kit (Invitrogen) according to the manufacturer's instructions except for dephosphorylation with calf intestinal phosphatase, which was omitted.
Number and activity of phagocytic cells from whole blood were determined using a commercially available kit (Phagotest®, Orpegen Pharma, Heidelberg, Germany) according to the manufacturer's instructions, except for the use of FITC-labeled S. aureus, instead of Escherichia coli.
DNA was extracted from 299 ticks and 86 bird-blood clots using DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer's instructions, except for an initial overnight incubation at 60°C with Proteinase K (Roche) at 1.44 µg/µl final concentration.
The running conditions for the focus gel is in accord to the manufacturer instructions, except for the Volt-hours which we set at 24 000 Volts- hour.
