Acidic phenol

Total RNA was isolated using acidic phenol (Sigma). Briefly, worms were washed off the plates using ice cold M9 buffer, followed by three additional washes. Next, 0.5 mL of AE buffer (acetic acid, EDTA), 0.1 mL of 10% SDS, and 0.5 mL of phenol were added and the mixture and vortexed vigorously for 1 min followed by incubation at 65°C for 4 min. The RNA was then purified by phenol:chloroform extraction followed by ethanol precipitation. The concentration and the purity of the RNA were determined by measuring the absorbance at 260/280 nm. To further determine the quality of the RNA, both the ribosomal 28 S and 18 S were visually inspected on an agarose gel. For Figure 4A and B, total RNA was purified from approximately 100 worms using Direct-zol^TM RNA MiniPrep kit (Zymo Research), as described by the manufacturer. cDNA was synthesized using total RNA and the SuperScript cDNA synthesis kit (Invitrogen, USA). Gene expression levels were then determined by real time PCR using the PowerSYBR® Green PCR Master Mix and StepOnePlus Real-Time PCR System (Applied Biosystems, USA). Relative gene expression was compared to actin as an internal control. Primers used are listed in Additional file 1: Table S7.

Total RNA isolation was carried out using TRIzol (Thermo-Fisher Scientific, Whaltman, MA, USA), as previously described [23 (link)]. All RNA samples used in this study were treated with DNase I (10 U of DNase I per 3 µg of total RNA; 37 °C for 1 h; in the presence of RNase inhibitor). After DNase I treatment, RNA samples were extracted with acidic phenol (Sigma Aldrich, St. Louis, MO, USA; pH 4.3) to eliminate any remaining traces of DNA. Taqman based qRT-PCR was performed using the one step Affymetrix HotStart-IT qRT-PCR Master Mix Kit (Affymetrix USB, Whaltman, MA, USA) and 50 ng of total RNA per reaction. Amplification conditions were 50 °C (10 min), 95 °C (2 min), followed by 40 cycles of 95 °C (15 s) and 60 °C (1 min). Target gene amplification was calculated using the formula 2^−∆∆Ct as described [23 (link)], primer and probe sequences are listed in Supplementary Table S1.

RNA isolation was performed using acidic phenol:bromochloropropane (BCP; Chomczynski and Mackey, 1995 (link); Gregorova et al., 2020 (link)). The cells were grown from the starter culture in 50ml of rMB until it reached OD₆₀₀ of 0.8. The cells were harvested at 3200g, 4°C for 10min and stored in −80°C. The pellet was resuspended in 4ml of 0.9% w/v NaCl solution followed by 4ml of acidic phenol (Sigma) and 800μl of BCP (Acros organic). Glass beads were added to break the bacterial cells and vortexed for 10min at room temperature (RT). The lysate was centrifuged at 10000g for 10min at RT, and aqueous phase was collected. The aqueous phase was re-extracted twice with phenol:BCP (2ml of acidic phenol and 400μl of BCP). Total RNA from the aqueous phase was precipitated by adding 2.5 vol of 99.6% v/v EtOH, at −20°C overnight and pelleted by centrifugation at 10000g for 20min at 4°C. The RNA pellets were air-dried and resuspended in RNAase-free ddH₂O water. RNA concentration was measured using a Nanodrop 2000 spectrophotometer (Thermo Scientific) and run on 2% w/v agarose, Tris-borate EDTA (TBE) gel with of Midori green (4μl per 100ml of 2% agarose) for quality assessment. The images were captured on Gel-doc XR (Biorad).

For each biological triplicate, 30 μg of total RNA was extracted from HEK293T cells transduced with lentivirus expressing WT or m⁶A-mutant reporters at a MOI of 3 and was fragmented to ∼100 nt fragments using 10x Ambion Fragmentation Reagent for 10 min at 75 °C. Fragments were immunoprecipitated with 5 μg of Rabbit anti-m⁶A antibody (Abcam) precoupled to 50 μl of Protein A/G magnetic beads (Pierce) in 300 μl of immunoprecipitation buffer (50 mM Tris–Cl pH 7.4, 150 mM NaCl, 1% NP-40 substitute, 0.5 mM EDTA) for 1 h at 4°C. Following three washes in IP buffer and in two washes in 0.25× SSPE, RNA was eluted twice with 125uL of elution buffer (50 mM Tris–Cl pH 7.4, 1 mM EDTA, 20 mM DTT, 0.1% SDS) by incubating at 42°C for 5 min. RNA was purified using acidic phenol (Sigma) and chloroform, and ethanol precipitated in the presence of 1 ul of GlycoBlue Coprecipitant (Ambion). RNA was finally resuspended in 10 μl of RNAse free water. cDNA was prepared with Superscript III and random hexamers (Invitrogen) and Real-Time PCR was performed using iTaq Universal SYBR Green Supermix in a CFX Connect Real-Time PCR system (Bio-Rad). To control for immunoprecipitation efficiency, the enrichment of WT and mutant reporters pulled down in each replicate was normalized to the enrichment of an endogenous m⁶A site found in ACTB.

Before the NRO reaction, NRO reaction buffer (10 mM Tris-Cl at pH 8.0, 5 mM MgCl₂, 1 mM DTT, 300 mM KCl, 50 µM ATP, GTP and Br-UTP, 2 µM CTP, 0.4 U/µL RNasin, and 2% sarkosyl) was prepared and preheated to 30°C for 5 min. An equal volume (100 µL) of NRO reaction buffer was mixed with 100 µL of thawed nuclei solution in freezing buffer and was incubated at 30°C for 5 min with mixing at 800 rpm on a thermomixer. Then, RQ1 DNaseI (Promega) was added along with DNaseI reaction buffer and samples were incubated at 37°C for 20 min with mixing at 800 rpm. To stop the NRO reaction, 225 µL NRO stop solution was added to the reaction and 25 µL of Proteinase K was added. The sample was incubated for 1 h at 55°C. Nuclear RNA was extracted with acidic phenol (Sigma) and then with chloroform (Sigma) and was precipitated and washed. RNA was then resuspended in 20 µL of nuclease-free water and subjected to base hydrolysis by addition of 5 µL of 1 N NaOH on ice for 10 min. The reaction was neutralized with 50 µL of 0.5 M Tris-Cl at pH 6.8. Then, RNA was purified through a Bio-Rad P-30 RNase-free spin column following the manufacturer's instructions and was treated with 7 µL of DNaseI buffer and 3 µL RQ1 DNaseI (Promega) for 10 min at 37°C and purified again with a Bio-Rad P-30 column.