RNA Detection

2. Isolate the RNA using the RNeasy kit following the manufac-
   turer’s protocol, being sure to use the gDNA Eliminator Col-
   umns to remove genomic DNA. Elute using 100μL of DEPC-
   treated nuclease-free water.


3. (Recommended): determine RNA integrity using an Agilent
   2100 Bioanalyzer or by agarose gel electrophoresis.


4. If desired, isolate the mRNA using the GenElute mRNA mini-
   prep kit (seeNote 3). Perform both the first and second elution
   using 50μL of DEPC-treated nuclease-free water rather than
   the supplied elution buffer, as the components of the elution
   buffer may interfere with downstream steps. Measure the con-
   centration of the mRNA via spectrophotometer.

3.2 RNA
Fragmentation,
Antibody Binding, and
Affinity Purification

Timing: Day 1—8 to 11 h + overnight incubation; Day 2—1 h

1. Adjust the volume of 1μg of polyA-enriched mRNA to 100μL
   in a 0.65 mL Bioruptor Pico Microtube (seeNote 4). Insert the
   tubes into the Bioruptor Pico sonication device prechilled to
   4 C. Fragment the RNA for 30 cycles of 30 s on/30 s off,
   which will produce fragments of 100–150 nucleotides.


2. (Recommended): verify RNA fragmentation using an Agilent
   2100 Bioanalyzer or by TBE gel electrophoresis (seeNote 5).


3. Save 5% (5 ng) of the fragmented mRNA as input at 80 C.


4. Make IP mixture (500μL per reaction): 950 ng (95μL) of
   fragmented mRNA, 100μLof5IP buffer, 12.5μLofm^6 A-
   specific antibody (0.5 mg/mL), 5μL of SUPERase inhibitor,
   and 287.5μL of DEPC-treated nuclease free water.


5. Rotate the IP mixture on a head-over-tail rotor at 4C for 2 h
   (seeNote 6).


6. Gently resuspend the Protein A beads using a vortex mixer.
   Wash 60μL of Protein A beads per reaction 3 times in ice-cold
   wash buffer using a magnetic rack. Resuspend the Protein A beads
   in 500μL of blocking buffer and rotate for 1 h (seeNote 7).


7. Remove the blocking buffer from the Protein A beads, and
   wash twice with 500μL of wash buffer. Remove the wash
   buffer, and add the IP mixture to the washed Protein A
   beads, and rotate the mixture on a head-over-tail rotor at
   4 C for 2 h.


8. Wash the beads–RNA mixture in wash buffer 3 times using a
   magnetic rack.


9. Add 50μL of elution buffer to the beads, and incubate the
   mixture for 1 h at 4C with continuous shaking (seeNote 8).


10. Remove and save the supernatant as eluent in a 1.5 mL low
    adhesion tube, keeping it on ice.


11. Repeatsteps 9and 10 and combine the two eluents.

Identifying the m^6 A Methylome 53