Recombinant ribonuclease inhibitor

Complementary DNA (cDNA) was reverse transcribed from total RNA using SuperScript III, RNaseOUT, Recombinant Ribonuclease Inhibitor, Random Primers and Oligo(dT)12-18 Primer (Thermo Fisher Scientific). Quantitative reverse transcriptase (qRT)-PCR was performed using the TaqMan Probe method. All primers and probe sets were designed by the Universal Probe Library Assay Design Center. All primers were synthesized by GeneNet, and all probes were purchased from Roche Diagnostics. The qRT-PCR assay was performed as previously described (Ishimoto et al., 2017) . Primer and probe sequences for each Custom TaqMan Gene Expression Assay are listed in Table S2.

RNA was extracted from cultured cells using the RNeasy Mini Kit (#74,106, Qiagen, Hilden, Germany) according to the protocol recommended by the manufacturer. Complementary DNA (cDNA) was reverse transcribed from total RNA using SuperScript III, RNaseOUT, Recombinant Ribonuclease Inhibitor, Random Primers and Oligo(dT)12-18 Primer (Thermo Fisher Scientific, Tokyo, Japan). Quantitative reverse transcriptase (qRT)-PCR was performed using the TaqMan probe method. mRNA expression was measured by qRT-PCR using the TaqMan probe (Roche Diagnostics, Basel, Switzerland), and the value for each gene was normalized to that for β-actin. mRNA expression was measured by qRT-PCR using the TaqMan probe (Roche Diagnostics, Basel, Switzerland), and the value for each gene was normalized to that for β-actin. All qRT-PCR experiments were performed using a LightCycler 480 System II (Roche Diagnostics). All qRT-PCR data are shown as the mean ± standard error of the mean. The MUC20 forward primer sequence was 5'-AGT GGG CAA AAC AAC TTC CTT-3′; the MUC20 reverse primer sequence was 5′-GCT TCC GAG GGG CTG TAG -3′ (Universal Probe Library Probes: 38#). The β-actin forward primer sequence was 5′-ATT GGC AAT GAG CGGTT-3′, and the β-actin reverse primer sequence was 5′-CGT GGA TGC CAC AGG ACT -3′ (Universal Probe Library Probes: #11).