Post-Transcriptional Methylation Removal in RNAs Using AlkB Demethylase and Subsequent RNA-cDNA Hybrid Hydrolysis in RNA Sequencing Preparation

To begin, prepare 10 milliliters of 2X AlkB reaction buffer and sterilize through a 0.2 micron syringe filter. In a sterile PCR tube, add 20 microliters of the linker 1-ligated RNAs, 50 microliters of the 2X AlkB reaction buffer, two microliters of the AlkB demethylase, one microliter of RNAs inhibitor, and 27 microliters of RNAs free water to prepare AlkB digestion reaction. Incubate the AlkB digestion mixture at room temperature for two hours to remove post-transcriptional methylation from the RNAs.

For clean phase separation, add 50 microliters of RNAs-free water into the AlkB reaction. Then, add 100 microliters of phenol, chloroform, and isoamyl alcohol mixture. Shake the tube for 10 seconds.

Centrifuge the AlkB mixture at 16, 000G for 10 minutes. Transfer approximately 140 microliters of the top aqueous layer-containing RNAs into a sterile 1.5 milliliter tube. To remove residual phenol, add 100 microliters of chloroform to the extracted RNAs and shake the tube.

Centrifuge the mixture at 16, 000G for 10 minutes and transfer approximately 120 microliters of the top aqueous layer into a sterile 1.5 milliliter tube. After reverse transcription reaction, add one microliter of five molar sodium hydroxide into the RNA cDNA hybrid. Incubate at 93 degrees Celsius for three minutes to hydrolyze the RNA strand of the RNA cDNA hybrid.

Then, add 0.77 microliters of five molar hydrochloric acid to neutralize the reaction. Prepare a 3%agarose gel in TAE buffer. Mix one microliter of 6X loading dye into five microliters of PCR product, and load the gel with the mixture.

Load five microliters of 50 or 100 base pair DNA ladders into the well before the first sample and the well after the last sample. Run gel electrophoresis at 120 volts and 400 milliamperes for 75 minutes to locate the PCR products. After electrophoresis, place the gel in a box and fill it with deionized water until the gel is completely immersed.

Add 10 microliters of ethidium bromide into the water. Wrap the box with foil, and stain for 30 minutes on a shaker. Discard the ethidium bromide-containing water into a waste bottle placed in a fume hood.

Rinse the gel with deionized water once and discard the water into a waste bottle. After washing the gel with deionized water for 10 minutes, use a gel imager to visualize the bands and acquire a high resolution image of the gel.