Rnase inhibitor

Mouse monoclonal anti-1-methyladenosine (clone AMA-2, IgG2b kappa subclass), anti-pseudouridine (clone APU-6, IgG1 kappa subclass), and anti-5-methylcytidine antibody (clone FMC-9, IgG2a lambda subclass) were established as described previously [7 (link), 16 (link), 21 (link), 22 (link)] and available for purchase from BML life science. Other antibodies and materials were purchased as follows: rabbit polyclonal anti-N6-methyladenosine antibody (Synaptic Systems), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Thermo), HRP-conjugated goat anti-rabbit IgG (Thermo), mouse IgG2a lambda (Sigma-Aldrich), DNase I (RNase-free, Qiagen), RNase inhibitor (Roche), sodium meta-arsenite (Wako), recombinant human FTO protein (Abcam), and recombinant human ALKBH5 protein (Abnova). The m⁶A-incorporated 15-mer ssRNA was synthesized and purified by HPLC (Gene Design) according to the sequence in the literature [10 (link)]. Human hepatocarcinoma HepG2 cells (HB-8065), human kidney HK-2 cells (CRL-2190), and human cervical carcinoma HeLa cells (CCL-2) were purchased from ATCC. E. coli (strain DH5α and HST04) was purchased from Takara-Bio. S. cerevisiae (strain BY4741 and W303-1B) was a kind gift from Prof. Shusuke Kuge (Tohoku Pharmaceutical University).

The rRNA was isolated as described [31 ]. The ethanol-precipitated pellets were resuspended in 200 μL RE-buffer (300 mM sodium-acetate; 0.5% (w/v) sodium dodecyl sulfate (SDS); 5 mM EDTA), supplemented with 8 Units RNAse Inhibitor (40 U/μL; Roche) and stored at 4 °C until use. Precipitated SDS was dissolved by gentle shaking at ambient temperature for 10 min. The ribosomal proteins were removed by successive phenol, phenol-chloroform-isoamylalcohol (25:24:1) and chloroform-isoamylalcohol (24:1) extractions. After a final centrifugation step at 15,000× g for 5 min, the RNA-containing solution (200 μL) was transferred to a new reaction tube and precipitated by adding 2 μL of glycogen (10 mg/mL) and 600 μL of a mixture of ethanol/0.3 M sodium acetate, pH 5. The rRNA was resuspended in 30 μL of DEPC-treated water and stored at −78 °C.

Knockdown of target genes using RNAi was basically preformed as described previously [24 (link)]. The siRNAs used here are listed in S2 Table. The RNAi efficiency was checked by real-time RT-qPCR with a set of primers listed in S2 Table. Total RNA (2 μg) extracted from the knockdown cells was treated with 2 U of RQ1 DNase (Promega) to remove genomic DNA in a 20 μl 1×Reaction Buffer (Promega) at 37°C for 30 min, followed by adding RQ1 DNase stop solution (Promega) and incubated at 65°C for 15 min. The DNase-treated total RNA (2 μg) was incubated at 65°C for 5 min in a 10 μL solution containing 2.5 μM oligo(dT18) primer, 60 μM random N6 primer, and 1 mM dNTPs, then cooled on ice. Subsequently, 10-μL mixture containing 2×Transcriptor RT reaction buffer (Roche), 10 U RNase inhibitor (Roche), and 5 U Transcriptor RTase (Roche) was added to the solution. The cDNAs were synthesized in the mixture by sequential incubation at 25°C for 10 min, at 55°C for 30 min, and at 85°C for 5 min. The PCR was performed in a 20 μL mixture containing a 1 μl aliquot of the cDNA solution, 0.2 μM of each PCR primers, and 1×KAPA SYBR FAST Master Mix optimized for LightCycler480(Kapa biosystems). The thermal cycling conditions included 45 cycles of 95°C for 10 s, 58°C for 20 s, and 72°C for 1 s. Amplification of cDNA was monitored by LightCycler 480 (Roche).

2 × 10⁷ RAW264.7 cells were left untreated or stimulated with 10 ng/ml LPS for 1 h, then harvested by scraping, washed twice with ice-cold PBS and lysed by repeated freeze-thawing in 1 ml of ice cold polysome lysis buffer (100 mM KCl, 10 mM HEPES [pH 7.0], 5 mM MGCl₂, 0.5% Nonidet P-40, 1 mM DTT, 100 U/ml RNase inhibitor, protease and phosphatase inhibitor cocktails [Roche]). Immunoprecipitations and mRNA measurements were essentially as described^{43 (link)}, except that a pre-immune (PI) rabbit serum was used as the immunoprecipitation control, and quantitative PCR was used to derive fold enrichment of various mRNAs in the anti-TTP immunoprecipitates compared with the PI controls (calculated as 2^−ΔCt).