Use RNase-free molecular biology-grade water for all buffers and RNase-free tips and tubes.
Materials and solutions needed
- Total RNA or mRNA preparation kit
- RNase-free molecular biology-grade water
- Fragmentation buffer 10x: 100 mM Tris-HCl pH 7.0, 100 mM ZnCl2
- 0.5 M EDTA
- 3M Sodium acetate, pH 5.2
- Ethanol, 100%
- Ethanol, 75%
- IP buffer 5x: 50 mM Tris-HCl pH 7.4, 750 mM NaCl and 0.5% (vol/vol) Igepal CA-630
- m6A Stock solution 20 mM: Dissolve 10 mg of m6A in 1.3 ml of molecular biology–grade, RNase-free water. Store aliquots of 150 µl at −20°C.
- Elution buffer: 1× Mix 90 µl (5× stock) of IP buffer, 150 µl (20 mM stock) of m6A, 7 µl of RNasin Plus and 203 µl of water (use molecular biology–grade, RNase-free water)
- m6A antibody stock solution: 0.5 mg/ml. Reconstitute 50 µg of lyophilized affinity-purified m6A-specific antibody in 100 µl of molecular biology–grade, RNase-free water.
- RNase inhibitor (e.g. RNasin)
- Nuclease inhibitor (e.g. ribonucleoside vanadyl complexes, RVC)
- Prepare total RNA or mRNA according to the kit manufacturer's instructions and adjust the RNA concentration to approx. 1 µg/µl with RNase-free water.
- Set up the fragmentation reaction in a thin-walled 200 µl PCR tube. 18 µl RNA solution, 2 µl 10x fragmentation buffer. Vortex and spin down the tube.
- Incubate the tubes at 94°C for 5 min in a preheated thermal cycler block with the heated lid closed. Remove the tubes from the block and immediately add 2 µl of 0.5 M EDTA. Vortex and spin down the tubes and place them on ice. Note: Stick to the specified amounts and volumes, as scaling may affect fragmentation efficiency and the resulting size distribution. If higher amounts are processed use several tubes.
- Collect contents of all tubes, add one-tenth volumes of 3 M sodium acetate, glycogen (100 µg/ml final concentration) and 2.5 volumes of 100% ethanol. Mix the contents and incubate at −80°C overnight.
- Centrifuge the tubes at 15,000 x g for 25 min at 4°C. Discard the supernatant. Do not to disrupt the pellet, which is easily visible because of the presence of glycogen. Wash the pellet with 1 ml of 75% ethanol and centrifuge again at 15,000 x g for 15 min at 4°C.
- Aspirate the supernatant and let the pellet air-dry. Resuspend the pellet in 300 µl of RNase-free water.
- Validate RNA postfragmentation size distribution by measuring RNA concentration with a spectrophotometer and running 0.5 µg of RNA on 1.5% (wt/vol) agarose gel for approx. 30 min. The outlined fragmentation procedure should produce a distribution of RNA fragment sizes centered around 100 nt.
- Save several µg of untreated fragmented RNA to serve as input control in RNA-seq.
- Adjust the volume of the remaining RNA to 755 µl with RNase-free water. Prepare the reaction mixture by adding 10 µl RNasin (200 - 400 U), 10 µl RVC (2 mM final concentration), 200 µl 5x IP buffer and 25 µl of m6A antibody (12.5 µg) to a final volume of 1 ml in a low-binding microcentrifuge tube. Vortex and spin down the tube. Set up a parallel reaction that includes the same amount of fragmented RNA but without the antibody. It will serve as a bead-only control to assess background levels and efficiency of RNA elution.
- Incubate with head-over-tail rotation for 2 h at 4°C.
- While the samples are incubating, wash 200 µl of protein A bead slurry twice in 1 ml of 1× IP buffer supplemented with RNasin and RVC. Resuspend the beads in 1 ml of 1× IP buffer supplemented with BSA (0.5 mg/ml), RNasin and RVC and incubate on a rotating wheel for 2 h. Spin down, remove and discard the supernatant and wash twice in 1 ml of 1× IP buffer supplemented with RNasin and RVC. Equally divide the beads between two 1.7 ml microcentrifuge tubes (one for the IP sample and one for the bead-only control).
- Transfer the IP-reactions into the bead-containing tubes prepared and incubate the reaction mixtures for 2 h head-over-tail at 4°C.
- Spin down the beads and carefully remove and retain the supernatant. Wash the beads with 1 ml of 1× IP buffer three times.
- Add 100 µl of elution buffer to the sedimented beads. Incubate the mixture for 1 h with continuous shaking at 4°C.
- Spin down the beads and carefully remove and retain the supernatant that contain the eluted RNA fragments.
- Add 100 µl of 1× IP buffer to the sedimented beads and gently tap the tube to mix. Spin down the beads and carefully remove and retain the supernatant (repeat steps 14 - 16).
- Combine all eluates from the same sample (IP or bead-only control) and add one-tenth volumes of 3 M sodium acetate (pH 5.2), and 2.5 volumes of 100% ethanol. Mix and incubate the sample at −80°C overnight.
- Centrifuge the tube at 15,000 x g for 25 min at 4°C. Discard the supernatant. Do not disrupt the pellet, which is not visible at the bottom of the tube. Wash the pellet with 1 ml of 75% (vol/vol) ethanol and centrifuge it again at 15,000 x g for 15 min at 4°C. Aspirate the supernatant and let the pellet air-dry. Resuspend the pellet in 15 µl of RNase-free water and quantify the RNA.
The RNA can now be used for library preparation.
For more background information and protocol details refer to Dominissini D et al., 2013.