Target specific forward and reverse primers

Reverse transcription (RT) was performed using the SuperScript II Reverse Transcriptase kit (Thermo Fisher Scientific), 0.5 mM of dNTP mix (Thermo Fisher Scientific) and 200 ng of random hexamer primers (Thermo Fisher Scientific). The cDNA was diluted 10x in water and stored at –20°C. 5 μl (37.5 ng) of the 10x diluted cDNA was mixed with 2× SYBR Select Master Mix (Thermo Fisher Scientific) and 200 nM of target-specific forward and reverse primers (Sigma-Aldrich) in a total volume of 10 μl. qPCR was performed with the QuantStudio 6 Flex machine (Applied Biosystems) with an annealing temperature of 60°C in a program using 40 cycles. Cycle threshold (Ct) values were used to calculate normalized expression (ΔΔCt method) and enrichment over input was calculated using the ΔCt method.

PCR mixtures containing 0.05 U/μl Ex Taq HS (Cat. No. RR006; TaKaRa Bio), 1 × Ex Taq buffer (Cat. No. RR006; TaKaRa Bio), 0.2 mM dNTP mixture (Cat. No. RR006; TaKaRa Bio), 0.5 μM Amplifluor UniPrimer II (Cat. No. S7905; Millipore), and 0.1 μM target-specific forward and reverse primers (Sigma Aldrich Japan) were prepared. Oligonucleotide sequences of target-specific primers are shown in Supplementary Table S1. Cells were resuspended in reagents for PCR in PCR tubes containing 1−2 × 10⁵ cells per 30 μl reaction. PCR was performed at 95°C for 10 min, followed by 50 cycles at 95°C for 10 s, 63°C for 20 s, and 72°C for 40 s, a final extension at 72°C for 5 min, and infinite hold at 4°C.