Sodium acetate

FAM-labeled Rhoa siRNA was synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). The sequences of negative control (NC) and Rhoa siRNA were as follows:
Rhoa siRNA sense: UGGCGGAUAUCGAGGUGGAUGGGAATT
Rhoa siRNA antisense: UUCCCAUCCACCUCGAUAUCCGCCATT
NC sense: UUCUCCGAACGUGUCACGUTT
NC antisense: ACGUGACACGUUCGGAGAATT
Chitosan (CS) was purchased from Solarbio, and its molecular weight (480~776 Da) was measured by matrix-assisted laser desorption time-of-flight mass spectrometry. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), arginine (Arg), hydroxylamine, sodium acetate, dimethyl sulfoxide, polyethylene glycol (PEG) 20,000, and deuterated heavy water were from Solarbio. NaAc sodium hydroxide, was from Macklin. Lipofectamine 2000 was from Invitrogen. Monoclonal antibodies to Rhoa and actin were from Cell Signaling Technology and Sangon Biotech (Shanghai, China) Co., Ltd., respectively.

Periplocoside E was extracted and purified from P. sepium Bunge (root barks) in the Institute of Pesticide Science, Northwest A & F University (NWAFU, Yangling, China) [37 ]. The chemical structure of PS-E is shown in Figure 1. PS-E has a purity of over 98% based on high performance liquid chromatography (HPLC) analysis. EAH sepharose 4B was purchased from general electronics (GE) Healthcare (Beijing, China). Tris, NaCl, HCl, acetic acid, sodium acetate, and all other chemicals were purchased from Solarbio (Beijing, China).

Quantitative real-time PCR (qPCR) analyses were performed to detect the relative abundance of the selected mRNA in the m⁶A antibody IP sample and in the input sample. Briefly, total RNAs were extracted from cells using the RNAiso plus reagent (Takara, Dalian, China). mRNAs were purified from total RNAs using the PolyATtract mRNA Isolation Systems (Promega, Z5310) and then fragmented using RNA Fragmentation reagent (Invitrogen, AM8740) for 1 min at 94°C. Protein A beads were washed and diluted into 500 μl IP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris, pH = 7.4, 100 U RNase inhibitor) and incubated with a m⁶A antibody (Synaptic Systems, 202003) for 1 h at 4°C. About 10% of the fragmented RNAs were left aside as input RNAs, whereas the remaining RNAs were added to the mixture and incubated for 4 h at 4°C with rotation. The mRNAs harboring m⁶A were eluted using 100 μl elution buffer (IP buffer, 6.7 mM m⁶A) for 1 h at 4°C and precipitated with 5 mg glycogen (Life Technologies, AM9510) and one-tenth volume of 3 M sodium acetate (Solarbio) in 2.5 volumes of 100% ethanol at −80°C overnight. The same numbers of the concentrated IP RNAs or input RNAs from each sample were used for cDNA synthesis. The m⁶A enrichment was finally determined by qPCR analysis.