Polyattract mrna isolation system

5’RACE-PCR was performed as previously described [37 (link)]. In brief, RNA was isolated with RNeasy mini/midi kit (Qiagen) according to the manufacturer’s protocol. Poly(A) containing RNA was purified from 500 μg of total RNA by affinity purification with biotinylated oligo-dT using PolyATtract mRNA Isolation Systems (Promega). 5’RACE PCR was performed using SMARTer 5’/3’ RACE (Clontech) according to the manufacturer’s protocol. PCR products were then run on 1% agarose gel, purified and cloned into pGEM-T Easy vector (Promega) and subsequently sequenced. Reverse transcription reaction for 5’RACE and qRT-PCR was performed using Superscript III Reverse Transcriptase (Invitrogen) using RNA extracted from 3 independent colonies. For qRT-PCR, transcript levels were normalized over gDNA to take into account differences in copy number between plasmids and normalized relative to act1⁺.

Total RNA was extracted from approximately 4 g of frozen tissues using the Large-Scale Column Plant RNAout kit (Tiandz, Inc., Beijing, China). To eliminate the remaining genomic DNA, the RNA was treated with DNase I (Takara, Dalian, China) according to the manufacturer's instructions. The integrity of the total RNA was assessed by running a 2 μl aliquot of the RNA sample on 1% formaldehyde denaturing agarose gel with ethidium bromide (EtBr). The concentration of RNA was estimated using a U-0080D spectrophotometer (Hitachi, Tokyo, Japan). Poly (A)⁺ RNA was isolated from 500 μg of total RNA with PolyATtract mRNA Isolation Systems (Promega, Madison, WI, USA). Double-stranded (ds) cDNA was synthesized from poly (A)⁺ using the SMART™ cDNA Library Construction Kit (Clontech, USA) according to the manufacturer's instructions and then purified using the QIAquick PCR Purification Kit (QIAGEN, Germany).

cDNA libraries were constructed using RNA Sample Preparation Kit (Illumia, San Diego, CA) following manufacturer's instructions. Briefly, mRNA was purified from total RNA using polyATtract mRNA isolation systems (Promega, Madison, WI). Fragmentation was carried out using divalent cations under elevated temperature in Illumina proprietary fragmentation buffer. First strand cDNA was synthesized using random oligonucleotides and SuperScript II (Promega, Madison, WI). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H (Promega, Madison, WI). Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. In order to select cDNA fragments of preferentially 200 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly). Products were quantified using the Agilent high sensitivity DNA assay on the Agilent Bioanalyzer 2100 system. Finally, the library preparations were sequenced on an Illumina Hiseq 2000 platform which generates 100 bp paired-end reads.

The tested compounds were freshly dissolved into cell media before feeding to HeLa cells at varying concentrations as indicated. After a 24 h incubation period, cells were harvested and the total cellular RNA was isolated with TRIZOL Reagent (Invitrogen). mRNA was extracted using PolyATtract^® mRNA Isolation Systems (Promega), followed by further removing of contaminated rRNA using RiboMinus Transcriptome Isolation Kit (Invitrogen). The isolated mRNA samples were digested by nuclease P1 and alkaline phosphatase following standard protocol. The nucleosides were separated by reverse phase ultra-performance liquid chromatography coupled with online triple-quadrupole LC mass spectrometer detection, and quantified by comparison with the standard curve obtained from nucleoside standards running the same batch of samples. The ratio of m⁶A/A or m⁶A/U was calculated.

Total RNA was extracted from porcine SPG, PS, and RS using TRIzol (Catalog No. 15596026, Invitrogen). mRNA was isolated using the PolyATtract mRNA Isolation Systems (Catalog No. Z5310, Promega, Madison, WI) following the manufacturer’s instructions. IP mixture was composed by 6 µg of rabbit anti-m⁶A antibody (Catalog No. 202003, Synaptic Systems), mRNA, IP buffer (50 mM Tris-HCl pH 7.4, 750 mM NaCl, and 0.5% NP-40), RNase inhibitor (Catalog No. AM2682, Invitrogen), and RNase-free water up to 500 µl in total volume. After being mixed by rotating for 2 h at 4 °C, the IP mixture was incubated with the Protein A beads (Catalog No. 10002D, Invitrogen) which have been washed for three times and blocked by 0.5 mg/ml BSA, followed by rotating overnight at 4 °C. Precipitated mRNA was eluted using elution buffer (1× IP buffer, 6.7 mM m⁶A). For the detection of the fold enrichment of m⁶A level, precipitated mRNA and input RNA were subjected to cDNA synthesis and qRT-PCR, respectively. The primers are listed in Table S3.

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.

Intact total RNA was extracted via centrifugation column (MiniBEST Universal RNA Extraction Kit; Takara) and mRNA was further purified via polyATtract mRNA Isolation Systems (Promega Corp.). Subsequently, m6A RNA immunoprecipitation (MeRIP) was performed with Magna MeRIP m6A kit (17–10,499, Millipore) according to the manufacturer’s instructions. The IP production was performed RT-qPCR as described above. The primers for RT-qPCR were showed in Table 1.