RNA Detection

12. Add 0.1 volumes (10μL) of 3 M sodium acetate to the com-
    bined eluents, followed by 2.5 volumes (275μL) of 100%
    ethanol. Mix well, and incubate the sample at 80 C over-
    night, or longer if desired (the RNA is stable at 80 C at this
    point). Do not add a glycogen carrier to the mixture, as it may
    interfere with downstream steps by precipitating free m^6 A
    (seeNote 9).


13. Centrifuge the tube at 16,000gat 4C for 25 min. Remove
    the supernatant, being careful to not disturb the pellet at the
    bottom of the tube. The pellet will not be visible.


14. Wash the pellet with 1 mL of 70% ethanol in DEPC-treated
    nuclease free water, and centrifuge the tube at 16,000gat
    4 C for 15 min. Remove all of the supernatant, being careful
    to not disturb the pellet at the bottom of the tube. Let the
    pellet air-dry on ice for 10 min, and resuspend the pellet with
    13 μL of DEPC-treated nuclease free water. Do not measure
    the RNA concentration, as it is too low to measure at this
    point.

3.3 Library
Preparation, Quality
Control, and
Sequencing

Timing: 1–2 days for library preparation and quality control; 5 days

for sequencing

1. Use 5μL of the affinity purified m^6 A-enriched RNA (“IP”) and
   2 μL of the input saved in Subheading 3.2 as starting material
   for mRNA library preparation. Transfer the sample to an 8-
   tube PCR strip. Add 3μL of DEPC-treated nuclease-free water
   to the input sample. Save the remaining mRNA as backup in
   case library preparation fails.


2. Add 12μLofFragment, Prime, Finish Mixfrom the Illumina
   TruSeq Stranded mRNA Library Preparation Kit to each sam-
   ple. Place the PCR strip containing the sample onto the ther-
   mal cycler, and run at 94C for 20 s, 4C hold, to prepare the
   RNA for first strand synthesis. Immediately proceed to the next
   step.


3. Perform first strand cDNA synthesis, second strand cDNA
   synthesis, adenylation, and adapter ligation according to the
   manufacturer’s instructions.


4. Measure the concentration of the cDNA library using qPCR:
   use 10μLof2Roche FastStart Essential DNA Green Master
   mix, 2μL of the Illumina PCR Primer Cocktail, 7μL of DEPC-
   treated nuclease-free water, and 1μL of the cDNA library.
   Determine the Ctat the midpoint of the qPCR amplification
   curve.


5. Enrich DNA fragments using the PCR kit provided in the
   TruSeq Stranded mRNA Library Preparation Kit. Use three
   fewer cycles than the Ct at the midpoint of the qPCR

54 Phillip J. Hsu and Chuan He