Any established RNA fragmentation protocol will work provided it results in RNA compatible with the application downstream of the enrichment. It is recommended that the RNA is cleaned up using spin columns or phenol chloroform-extraction and ethanol precipitation post-fragmentation.
1) Make your own RNA fragmentation buffer, which at 1X is composed of:
- 40 mM Tris-OAc
- 100 mM KOAc
- 30 mM Mg(OAc)2
- pH 8.3 @ 25°C
The Stop Solution is 0.5M EDTA (pH 8.0).
2) If you intend to proceed directly to NGS library preparation, you can also fragment in the NEBNext First Strand Synthesis Reaction Buffer provided with NEB #E7771, then proceed to first strand synthesis. Please note that fragmentation is slightly slower in this buffer.
