Primescript rt reagent kit and gdna eraser

We used RNAiso Plus (TaKaRa, Tokyo, Japan) to extract the total RNA from the tissues. The quality of RNA was detected by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). Next, reverse transcription was performed with T100™ thermal cycler (BIO-RAD, US) using PrimeScript^TMRT reagent kit and gDNA eraser (TaKaRa, Tokyo, Japan). Quantitative real-time PCR (qPCR) was performed in LightCycler^® 96 (Roche, Switzerland) with TB Green®Premix Ex Taq™ Ⅱ (Tli RNase H Plus) (TaKaRa, Tokyo, Japan). Primer sequences are listed in Table 1.

Total RNA was isolated with TRIzol, and cDNA was synthesized with the PrimeScriptTM RT Reagent Kit and gDNA Eraser (TaKaRa, Cat No. RR037A). Real‐time PCR was performed using the SYBR Green PCR Mastermix (Solarbio, Cat No. SR1120) and a Rotor‐Gene‐Q instrument (Qiagen). The fold change in target expression between WT and KO mice was calculated using the 2^−ΔΔCt method,²⁹ and GAPDH was used as an internal reference. The primers are shown in Table S2.

Nuclear/cytoplasmic fractionation of MuSCs was performed using a cytoplasmic/nuclear RNA purification kit (Norgen Biotek, Thorold, ON, Canada) according to the manufacturer’s instructions. Briefly, MuSCs (seeded at ~2 × 10⁷ cells per dish) cultured on a 10-cm culture dish were washed twice using cold PBS after 3 days of differentiation and were lysed in a hypotonic buffer (10 mM Tris pH 8.0, 1 mM EDTA). To separate nuclei and debris, cells were centrifuged at 500× g for 5 min. Total RNA was extracted separately from the supernatant (cytosolic fraction) and the pellet (nuclei) using RNAiso Plus reagent (TaKaRa, Dalian, China) and reverse-transcribed into cDNA using the PrimeScript^TM RT Reagent Kit and gDNA Eraser (Takara, Dalian, China). RNA was quantified by qRT-PCR. 18S ribosomal RNA (18S rRNA) and U6 were used as cytoplasmic and nuclear controls, respectively. All procedures were performed at 4 °C under RNase-free conditions.