Molecular Biology Techniques

Primers for PCR reactions were designed using Primer3 v.0.4.0 (Koressaar and Remm, 2007; Untergasser et al., 2012), and purchased from Hy Laboratories (Rehovot, Israel). Primers used in PCR and qRT-PCR reactions are described in Supplementary Tables S1, S3, respectively. PCR reactions were performed in an Eppendorf (Hamburg, Germany) Thermal Cycler using REDTaq ready mix (Sigma-Aldrich) in 25-μl reaction volumes, according to manufacturers’ instructions. Unless stated otherwise, PCR products were separated by gel electrophoresis at 120 V for 30 min on a 1% agarose gel in 0.5X Tris-acetate ethylenediaminetetraacetic acid (EDTA) buffer. Gels were stained with 1 μg/ml ethidium bromide and gel images were captured using a C200 gel imaging workstation (Azure Biosystems, Dublin, CA, United States). PCR bands were extracted with the AccuPrep PCR purification kit (Bioneer, Daejeon, South Korea) and sent for sequencing at Hy Laboratories. qRT-PCR reactions were carried out in a LightCycler 480 System (Roche, Basel, Switzerland) with 5xHOT FIREPol^® EvaGreen^® qPCR Mix Plus (no ROX) (Solis BioDyne, Tartu, Estonia). qPCR reactions were carried out in 10-μl reaction volumes that contained 2 μl of qPCR mix, 0.25 μl of forward primer, 0.25 μl of reverse primer (10 pmol/μl), 2.5 μl of DNA and 5 μl of nuclease-free water. Restriction enzymes were purchased from New England Biolabs (Ipswich, MA, United States).