qRT-PCR and semi-quantitative RT-PCR

1 μg of Turbo DNase-treated RNA was reverse transcribed with the iScript cDNA synthesis kit (BioRad, 1708890), treated with 0.5 μL RNase cocktail (Invitrogen, AM2286), and diluted 1:10. cDNA was quantified using the SensiFAST SYBR No-Rox kit (Bioline, BIO-98005) and 1 μM of each primer (primer sequences provided in Supplementary Table ⁵). qPCR settings were as follows: 95 °C for 10 min and 40 cycles consisting of 10 s at 95 °C, 20 s at 60 °C, and 20 s at 72 °C, followed by melting curve analysis. TER1 and U6 levels were normalized to act1 mRNA levels and the average wild-type Ct value, and subsequently subject to unpaired two-tailed Student’s t tests and, where applicable, one-way ANOVA followed by a Tukey post hoc test with α set to 0.05 (Supplementary Data ⁴).

For semi-quantitative RT-PCR, DNase-treated RNA, 10 nmol dNTP mix, and 10 pmol gene-specific reverse primers (Supplementary Table ⁵) were heated to 65 °C and slow-cooled to 37 °C before reverse transcription with 5 U AMV-RT (NEB, M0277L) at 42 °C for 1 h. cDNA was amplified with Taq polymerase (NEB, MO273L) using standard protocols and the following cycling conditions: 5 min initial denaturation at 94 °C, 22 (TER1) or 17 (U6) cycles of 30 s at 94 °C, 30 s at 57 °C, and 1 min at 72 °C, and a final 10 min extension at 72 °C.