For dephosphorylation of RNA 3'ends, pH 6.5 PNK buffer is used, rather than the pH 7.6 buffer, provided by NEB. Have you compared these two conditions internally? Is 5x PNK pH 6.5 buffer available commercially?
This buffer needs to be prepared by the user. We haven’t compared conditions, but increased phophatase activity of PNK at lower pH has been reported in literature, you can read more in (Wang and Shuman, 2002).
For the RNA ligation step, do you think PEG4000 works as well as PEG400?
PEG4000 works fine in solution, but when used on-bead, it in our hands interfered with immunoprecipitation efficiency. We are not sure why, but it might have a greater tendency to stick to the beads.
Is it possible to use a 3' linker with a phosphorylated 5' end instead of a pre-adenylated 5' end and adding some ATP during the 3' linker ligation step?
Yes, just follow the protocol as described in (König et al., 2010). It is important in this case to also dephosphorylate using a phosphatase (as described in original protocol), rather than PNK, because contaminating PNK in the ligation reaction containing ATP would lead to phosphorylation of 5’ ends of RNA, and their self-circularisation.
Why do you use final 10mM DTT in the ligation buffer, even though NEB uses 1mM final DTT? And why do you make 4x stock of buffer?
Several companies use 10mM final concentration (including Ambion, Takara and Promega), and this concentration was tested in our hands. DTT also inhibits RNases, therefore we decided to use the higher concentration. Furthermore, DTT is unstable, therefore higher concentration ensures that enough reducing activity is present during overnight incubation. We make 4x stock of buffer to avoid precipitation, which can occur with 10x stock.
32P gamma ATP labelling
You say to use 0.8 uL of P32 gamma ATP. what concentration of mCi/unit should I use?