# Downregulation of Squalene Synthase Broadly Impacts Isoprenoid Biosynthesis in Guayule
## 4 Materials and Methods
### Genomic DNA and RNA Extractions and PCR and qPCR Analyses  
Plant DNA was extracted using a Sigma GenElute Plant Genomic DNA Miniprep Kit (Sigma-Aldrich, Carlsbad, CA, USA). Approximately 150 mg leaf tissue was cut from plants grown in tissue-culture, placed into 2 mL tubes, and snap-frozen in liquid nitrogen. A bead was added to pulverize the tissues into a fine powder at a frequency of 30/s for 1 min using the mixer mill MM 400 tissue lyser (Verder Scientific, Inc., Newtown, PA, USA). PCR was carried out in 50 uL of a mixture containing GoTaq Green Master Mix (Promega Corp., Madison, WI, USA), 200 ng guayule genomic DNA or 20 pg plasmid DNA, and 1 uM of _BAR_-specific primers:  
5\({}^{\prime}\)-ATGACGCCAAGACGCCCGCC-3\({}^{\prime}\) and 5\({}^{\prime}\)-GATCTCGGTGACGGGCAGGCG-3\({}^{\prime}\). After heating the samples to 95 \({}^{\circ}\)C for 2 min, the reaction proceeded with 40 cycles of 95 \({}^{\circ}\)C for 30 s, 71 \({}^{\circ}\)C to amplify the product \(\sim\)0.5 kb in the _SQSi_ lines for 30 s, and 72 \({}^{\circ}\)C for 1 min. A final elongation step was carried out at 72 \({}^{\circ}\)C for 5 min. PCR products were separated by electrophoresis on a 1% (\(w/v\)) agarose gel.  
The iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) was used to synthesize complementary DNA (cDNA) for both RT-PCR gel electrophoresis analysis and quantitative RT-PCR, following manufacture instructions. RNA extraction, cDNA synthesis, and qPCR analysis were performed as previously described [71], with the following changes: for qPCR analysis, cDNAs were diluted 1:10 in 15 uL volume reactions con taining iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). Thermocycler 7500 Fast Real-Time PCR system (Applied Biosystem, Foster City, CA, USA) conditions: 95 \({}^{\circ}\)C preincubation for 3 min; amplification for 40 cycles at 95 \({}^{\circ}\)C for 15 s and 60 \({}^{\circ}\)C for 30 s; the dissociation stage for 95 \({}^{\circ}\)C for 15 s, 60 \({}^{\circ}\)C for 1 min, and 95 \({}^{\circ}\)C for 15 s. Each qPCR run was performed with three independent tissue samples, each sample having two technical replicates. The 185 gene was used as an internal control with the following respective forward and reverse primer sequences: 5\({}^{\prime}\)-CAACAACCCCGACTTTCCG-3\({}^{\prime}\)/5\({}^{\prime}\)-CACCGTACCACCATAGTA-3\({}^{\prime}\). Target genes forward/reverse primer pair sequences are: _SQS_, 5\({}^{\prime}\)-GATCCCAATGCCCAACTACAA-3\({}^{\prime}\)/5\({}^{\prime}\)-CCAGAGGCCTTGCATATTTTCTG-3\({}^{\prime}\); _HMGR_, 5\({}^{\prime}\)-ATGGCATTTTCTGGAACATTG-3\({}^{\prime}\)/5\({}^{\prime}\)-CTCTCCGCCCTTCTATCC-3\({}^{\prime}\); _MVK_, 5\({}^{\prime}\)-GGCATCCTGTGCTATGACA-3\({}^{\prime}\)/5\({}^{\prime}\)-GCATTTGAGCAAACCTTGT-3\({}^{\prime}\); _FPPS_, 5\({}^{\prime}\)-TCAACGATCCGCCTTCG-3\({}^{\prime}\)/5\({}^{\prime}\)-TCCAGGTTGTGTGCAAGCA-3\({}^{\prime}\); _CPT_, 5\({}^{\prime}\)-GCTTTTTTCGGGCATTTCA-3\({}^{\prime}\)/5\({}^{\prime}\)-TGCCAAGAATCCGCTTTT-3\({}^{\prime}\); _CBP_, 5\({}^{\prime}\)-GGCG TCATATGAGAGA-3\({}^{\prime}\)/5\({}^{\prime}\)-GATTGCTACTGCATATATG-3\({}^{\prime}\); and _SRPP_, 5\({}^{\prime}\)-GTGGC CAACACATGTACGTAAA-3\({}^{\prime}\)/5\({}^{\prime}\)-TTCTCAGCTACCGGCTCGTAGT-3\({}^{\prime}\).