Quantstudio 3 instrument

Livers were harvested from female CYP3A(−/−), FVB wild-type, OATP1B2(−/−), and DBA wild-type mice between 8 and 12 weeks of age. Livers were snap frozen and stored at −80 °C until RNA isolation. RNA was isolated by utilizing an E.Z.N.A. Total RNA Kit I (Omega Bio-tek, Norcross, GA, USA), followed by cDNA synthesis with qScript XLT cDNA SuperMix (Quantabio, Beverly, MA, USA). Real-time polymerase chain reaction (qPCR) was performed using TaqMan probes for OATP1B2 (Mm00451513_m1), CYP3A11 (Mm00731567_m1), and GAPDH (Mm99999915_g1) (Thermo Fisher Scientific) and a QuantStudio 3 instrument (Thermo Fisher Scientific).

For RT-qPCR, total RNAs were extracted from indicated tissues using RNAiso Plus (Takara, 9109) as per the manufacturer’s guidelines. Following the removal of residual genomic DNA with Turbo DNase (Invitrogen), 500 ng of total RNA was reverse-transcribed into cDNAs using the PrimeScript RT Reagent Kit (Takara, RR037A). RT-qPCR was performed on a QuantStudio 3 instrument (Thermo Fisher) utilizing a SYBR Premix Ex Taq kit (Takara, RR820A). Relative gene expression was analyzed based on the 2^−ΔΔCt method with β-actin as an internal control. For RIP assay of MIWI- or MILI-associated piRNAs, total RNAs were extracted from anti-MIWI or -MILI IP pellets and then labeled with [γ-³²P]-ATP (PerkinElmer) by T4 PNK (Thermo Fisher, EK0032) after FastAP (Thermo Fisher, EF0651) treatment. They were resolved on a 15%, 7 M urea polyacrylamide gel along with radioactive oligoribonucleotide size markers, followed by autoradiography. All primers used for RT-qPCR are listed in Supplementary Table 1.

Total RNA was extracted by Trizol reagent (Thermo Fisher, Waltham, MA) following the manufacturer’s instructions. Total RNA (2 µg) was used to perform reverse transcription with Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher). Quantitative real-time RT-PCR analysis was performed with a Quantstudio3 instrument (Thermo Fisher) using Fast Start Universal SYBR Green Mix (Roche, Mannheim, Germany). The primers, synthesized by Sangon Biotech., Shanghai, China., and used for real-time RT-PCR were displayed in Table S2. The method of 2^−∆∆Ct was used to calculate the relative mRNA level. Data were normalized to GAPDH expression.

Total RNA was extracted using TRIzol reagent and was treated with RQ1 RNase-Free DNase (Promega, Madison, WI, USA) for 30 min. cDNA was synthesized using the M-MLV reverse transcription kit (Promega) according to the manufacturer’s instructions. qPCR was conducted using ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech, Nanjing, China) on a QuantStudio 3 instrument (Thermo Fisher Scientific). The mRNA level of the β-actin housekeeping gene served as a control. qPCR primer sequences are listed in Additional file 1: Table S1.

For qPCR first-strand cDNA synthesis, 2 µg of RNA was reversed-transcribed using High-Capacity RNA-to-DNA™ reagents (Applied Biosystems, Waltham, MA, USA). EXPRESS SYBR™ GreenER™ Supermix with ROX, or specific TaqMan assays (ThermoFisher, Waltham, MA, USA), were used for qPCR reactions; all following the manufacturer’s instructions. Primer/probe sequences were reported previously [17 (link)] or are reported in the supplementary materials (Supplementary Materials Table S3). H19, miR-675-3p and miR-675-5p qPCR assays used primer/probe sets from GeneCopoeia (Rockville, MD, USA). A QuantStudio 3 instrument (ThermoFisher, Waltham, MA, USA) was used for sequence detection. qPCR assays were run in duplicate, with relative expression calculated by the 2^−ΔΔCt method. All assays were normalized to 18S rRNA, except for miR-675 RNAs, which used U6 RNA.