Rnase r enzyme

Cytoplasmic RNA was isolated with RNeasy Mini Kit (Qiagen, Germany) and the remaining nuclear RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Nuclear and cytoplasmic protein fractionation was performed using the Nuclear and Cytoplasmic Extraction Kit (Vazyme, Nanjing, China). For RNase R treatment, 2 μg total RNA was incubated with 6 U RNase R enzyme in 1× buffer (Epicentre, USA) for 30 min at 37 °C. RNA was purified using the ethanol precipitation method according to the standard protocol. For actinomycin D treatment, MGC-803 cells (2 × 10⁵/well) were seeded in 6-well plates overnight and treated with 2 mg/mL actinomycin D or DMSO control (Sigma-Aldrich, USA) for 0 h, 4 h, 8 h, 12 h, 18 h and 24 h. Total RNA was then isolated for RT-qPCR analysis.

Adult brain samples (sample 1–5, Supplementary Materials) were obtained from the Harvard Brain Tissue Resource. Total RNA was extracted from 5 adult frontal lobe samples. To obtain total RNA, tissue pieces were first fixed in OCT and two sections were used for total RNA purification. Total RNA purification for these samples and 6 human blood samples was performed using RiboPureTM RNA purification kit (Ambion) according to the manufacturer instructions. Total RNA from fetal and adult liver, fetal and adult heart, and placenta were purchased from Clonetech. Total RNA from the FFPE tissues was extracted using High Pure FFPET RNA isolation kit (Roche) according to the manufacturer instructions. RNA from the serum was purified using Plasma/Serum Circulating and Exosomal RNA purification Kit (Norgen). RNase R treatment of total RNA was performed after ribosomal RNA depletion. Briefly, 2 μg RNA was added to a mixture of 1x RNase R buffer (Epicentre®), 1 mM MgCl_2, and 1.0 μl (20 U/μl) RNase R enzyme (Epicentre®). The mix was then incubated at 37 C° for 10 minutes. RNase R was then inactivated by adding 1:1 volume of H₂O. All samples are listed in Supplementary Materials.