Protocol FAQ

Why the 3' adaptor ligation contains DTT in input samples while not in the CLIP samples? Does this have a relationship to the fact that DTT breaks antibody disulfite bond? If so, why was the DTT used in PNK treatment?

Yes – some antibody-antigen interactions seem particularly sensitive to DTT, and for the RNA adapter ligation our experience was that it didn’t make a significant enough impact to the adapter ligation to be worth including in that step. For the PNK it seemed to be more helpful, though we’re currently working on some improved methods that drop the DTT in the PNK step as well

It seems that the ATP is needed for 5’ phosphorylation, why does your reaction doesn't contain ATP? Is there any particular reason that the buffer pH for the PNK is 6.5?

The PNK step is not for 5’ phosphorylation, it’s to remove 2’-3’ cyclic phosphates left by RNase I. That activity of PNK seems to be most efficient at acidic pH (there’s a paper or product note or something that shows this activity in different buffers that I’m having trouble finding right now, but that acidic PNK step has been common in pretty much all CLIP protocols that use RNase I or other RNases that leave the 2’-3’ cyclic phosphate)