Dntp mix

Each single CTC was transferred to 0.2 ml PCR tube and subjected to lysis and RNA extraction according to the manufacturer’s specifications (Single Cell Lysis Kit, Thermo Fisher Scientific, MA, USA). 2.5 μM oligo (dT) primers and 0.5 mM dNTP Mix (all Life Technologies, Singapore) were added into the lysed CTC sample, which was subsequently incubated at 65°C for 5 min and cooled on ice for at least 1 min. 1x first-strand buffer, 5 mM DTT, 10 U RNaseOUT Recombinant RNase Inhibitor, and 50 U SuperScript III RT (all Life Technologies, Singapore) were used, made up to a final volume of 20 μl in nuclease-free water. The final product was incubated at 25°C for 5 min, 55°C for 60 min, and 85°C for 5 min for reverse transcription on a C1000TM Thermal Cycler (Bio-Rad, Hercules, USA).

In total, 1 μl of 50 μM oligo (dT) ₂₀ primers (Life Technologies) and 1 μl of 10 mM dNTP Mix (Life Technologies) were added to 5 μl of each lysed RNA sample. The sample was incubated at 65 °C for 5 min and subsequently cooled on ice for at least 1 min. In all, 1 × first-strand buffer, 5 mM DTT, 40 U RNaseOUT Recombinant RNase Inhibitor, 200 U SuperScript III RT (all Life Technologies) were added to a final volume of 20 μl. The following thermal setting was applied on a Verity 96-well Thermal Cycler (Applied Biosystems): 25 °C for 5 min, 55 °C for 60 min and 85 °C for 5 min. Each RT product was diluted to a final volume of 40 μl to avoid qPCR inhibition.

Total RNA was purified from HDMECs and HeLa cells using the RNeasy kit, according to the manufacturer’s instructions (Qiagen, Manchester, UK). RNA quantity and quality were determined using a NanoDrop 1000 Spectrometer (Thermo Fisher Scientific). Then, 1 µg of RNA was reverse-transcribed to cDNA using the reverse transcriptase Superscript III in addition to oligo dT, random hexamers, dNTP mix, and RiboLock (Life Technologies, Paisley, UK). To set up a 25 µL reaction for qPCR, 10ng of cDNA was mixed with RNase free water, 2× Power SYBR Green (Life Technologies, Paisley, UK) and forward and reverse primers to a final concentration of 400 nM (Life Technologies, Paisley, UK).
Primer sequences (5’ to 3’):
β-ACTIN Fwd GATGAGATTGGCATGGCTTT; β-ACTIN Rev CACCTTCACCGTTCCAGTTT. MEF2A Fwd CAGGGAGTTCACTGGTGTCC; MEF2A Rev CTTGGTGGTCTCTGAGGAGC. MEF2B Fwd GTTCACCAAGCGGAAGTTCG; MEF2B Rev GCATACTGGAAGAGGCGGTT. MEF2C Fwd CGAGATGCCAGTCTCCATCC; MEF2C Rev GTGAGCCAGTGGCAATAGGT. MEF2D Fwd AGGAAGAAGGGCTTCAACGG; MEF2D Rev GTCGGTACTTGTCCTCCAGC.
qPCR was performed on an ABI ViiA 7 Thermocycler (Applied Biosystems) with the following parameters: 50 °C for 2 min; 95 °C for 10 min; (95 °C for 15 s and 60 °C for 1 min) × 40 cycles. For each sample, the average cycle threshold (CT) value was normalised to β-ACTIN and then compared to the relevant control sample using the comparative CT (2-ΔΔCT) method.

RNA was extracted from cells using Trizol (Life Technologies) and purified using the RNeasy kit (QIAGEN). Reverse transcription was performed using Superscript III (Life Technologies) according to manufacturer’s instructions. Polymerase chain reactions (PCR) were set up as follows: cDNA (25 ng), AmpliTaq Gold 10x buffer (Applied Biosystems), MgCl₂ (1.5 mM), forward and reverse primers (1 µM each), dNTP mix (0.5 mM; Life Technologies) and 1 U Taq polymerase (AmpliTaq Gold, Applied Biosystems). Primer sequences are detailed in Table S1.