Qiacube automated

Genomic DNA was isolated from whole blood of patients enrolled in the IFIGEN study using the QIAcube automated system (Qiagen). SNPs were selected based on their ability to tag surrounding variants with a pairwise correlation coefficient r² of at least 0.80 and a minor allele frequency ≥5% using publically available sequencing data from the Pilot 1 of the 1000 Genomes Project for the CEU population. Supplementary Table 3 reports sequences of the primers used in the study. Genotyping was performed using KASPar assays (LGC Genomics) in an Applied Biosystems 7500 Fast Real-Time PCR system (Thermo Fisher Scientific), according to the manufacturer’s instructions. Mean call rate for the SNPs was > 98%. Quality control for the genotyping results was achieved with negative controls and randomly selected samples with known genotypes.

DNA was extracted from 20-μm-section paraffin embedded tumor samples after a 24 h digestion by proteinase K, using the DNeasy Tissue Kit and the QIAcube automated extractor (Qiagen, Hilden, Germany). Yield and quality of DNA were evaluated by Qubit fluorometer (Invitrogen, Carlsbad, CA). Direct Sanger sequencing for mutational assessment of exon 15 of BRAF was performed following PCR amplification as previously described [14 (link)]. PCR was carried out on 20 ng of DNA in 10 μl final volume and 1 U of Hot Start Taq polymerase (Qiagen). The amplified products were studied by direct sequencing after clean-up exonuclease ExoSAP-IT (Affymetrix, Santa Clara, CA) using the Big Dye Terminator Cycle Sequencing Kit and capillary electrophoresis on the automated sequencer ABI3730 (Applied Biosystems, Carlsbad, CA). Sense and antisense sequences were screened for exonic alterations using SeqScape v2.5 software (Applied Biosystems) and compared with the NCBI reference sequences: BRAF (NM_004333.4).

DNA was extracted using the QIAamp DNA Blood Mini kit 250 (Qiagen, Hilden, Germany) according to the manufacturer’s instructions on the QIACube automated nucleic acid extraction system (Qiagen). Samples were quantified by Nanodrop spectrophotometry or Qubit DNA analysis (Thermo Fisher Scientific, Waltham, MA, USA).

Hypocotyls of 21-days-old seedlings and the third internodes of 35-days-old seedlings grown under low or normal light intensity were harvested, respectively. Total RNAs were extracted using RNeasy Plant Mini Kit on the QIAcube automated system (Qiagen), followed by reverse-transcribed using a ReverTra Ace qPCR RT Master Mix with gDNA remover (TOYOBO) following the manufacturer’s instructions. Quantitative PCR was performed with GoTaq qPCR Mater Mix (Promega) on LightCycler 96 (Roche). The expression levels of SlIAA3 (5'-GCAATTCAAATCAATCATTCTTTCT-3' and 5'-GTTTATTATCCCAGGCAAACCTAAT-3') were normalized relative to those of a reference gene, Slactin7-like (5'-TGTCCCTATCTACGAGGGTTATGC-3' and 5'-AGTTAAATCACGACCAGCAAGAT-3'; Hashimoto et al., 2018 (link)). Relative expression was calculated by two to the power of relative expression (log₂), calculated by subtracting Ct values of IAA3 from those of Slactin7-like.