Sybr green universal master mix

qRT-PCR was performed using the StepOnePlus Real-Time PCR System (Life Technologies). Four microliters of DNA was used for each of the three technical repeats for each of the three biological repeats. The DNA was amplified using SYBR Green Universal Master Mix (Life Technologies) and the primers described in Supplementary Table S3. The data were analyzed using the ΔΔCt method for PKL-cMYC and the ΔCt method for H3K4me3. The mean relative fold changes or ΔCt values, SEM and statistical significance values are found in Supplementary Tables S14 and S15.

Thyroid tissue samples were collected during surgery, immediately placed in RNA later solution, and stored at −80 °C for further use. Total RNA and DNA were extracted from RNA later-preserved tissue derived from paired tumors and normal samples from the same patient. Tissues were homogenized and followed by lysis with TissueLyser III (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer’s protocol. Nucleic acids were purified with the AllPrep DNA/RNA Mini Kit (Qiagen, Inc., Valencia, CA, USA). DNA and RNA concentrations were measured with a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Reverse transcription was performed using a SuperScript IV VILO Master Mix (Invitrogen Life Technology, Carlsbad, CA, USA) including no-RT control reactions for each experiment. The RT² qPCR Primer Assay (Qiagen, Inc., Valencia, CA, USA) for KDM5C (NM_004187), KDM5D (NM_004653 and KDM6A (NM_021140), and 18S (NR_003286) was used for gene expression analyses by real-time PCR. Each sample was run in triplicate using a LightCycler^® 96 (Roche, New York, NY, USA) with a SYBR™ Green Universal Master Mix (ThermoFisher Scientific, Inc., Waltham, MA, USA). 18S (NR_003286) was used as a control for the amplification of target genes by real-time PCR. The relative fold gene expression of samples was calculated using the delta-delta Ct method.

The mutational status of BRAF^V600 (exon 15) was determined as previously described^{62 (link)}, whereas the β-catenin^A121G point mutation was verified by allele-specific quantitative PCR (qPCR). Forward primer (5′-TTGATGGAGTTGGACATGGC-3′) was common to wild type (wt) and mutated (mut) sequences, whereas two different reverse primers with substitution of a single base at the end of the primer (reverse wt 5′-TCAGAGAAGGAGCTGTGGT-3′ and reverse mut 5′-TCAGAGAAGGAGCTGTGGG-3′) were designed to amplify wt or mut allele, respectively. QPCR reactions for wt and mut alleles were run in parallel on 10 ng of genomic DNA in a final volume of 20 μL SYBR-Green Universal Master Mix (ThermoFisher Scientific) at 95 °C for 10 min, followed by 45 cycles at 95 °C for 15 s and at 60 °C for 1 min, and dissociation performed at 95 °C for 15 s, 60 °C for 20 s, and 95 °C for 15. Known amounts of wt and mut DNA molecules were used to generate absolute standard curves. The copy number of wt and mut alleles was established by extrapolation from the standard curves. The percentage of mut allele was defined as the ratio between mut DNA molecules and the sum of wt and mut DNA molecules. The mutation within splicing site at the exon–intron 9 junction of TP53 gene was verified by Sanger sequencing using the following primers: TP53_forw, 5′-ACTGCCCAACAACACCAGCTCCT-3′ and TP53_rev, 5′-CATCACTGCCCCCTGATGGCAAA-3′.

The IQGAP1-knockdown was performed according to the protocol reported in our previous study, with a few modifications based on Zoheir et al. (24 (link)). The IQGAP1 sequence (NC_000015.10) was obtained from the NCBI gene bank.
Diethylnitrosamine (DENA) was purchased from Sigma Chemicals (St. Louis, MO, USA). The TRIzol reagent was purchased from Invitrogen (Carlsbad, CA, USA), while the high-capacity cDNA reverse transcription kit, the primers for real-time PCR, and the SYBR Green Universal Master mix were all bought from Applied Biosystems^® (Foster City, CA, USA). Antibodies specific for b-actin, IQGAP1, and p53 were purchased from Santa Cruz Biotechnology.

Tail snip DNA was extracted using KAPA Express Extract Kit (Roche). Genotyping PCR was performed with HotStarTaq Master Mix (Qiagen) according to manufacturer’s instruction. Genotyping primers used are summarized in Table S3. To determine the zygosity of HSC-Scl-CreER^T, qPCR was additionally performed with purified tail snip DNA using SYBR^™ Green Universal Master Mix (Applied Biosystems).