iiCLIP protocol

Questions pertaining to iiCLIP protocol answered by Flora Lee.

I have a couple of questions for you regarding the 3' adapter ligation step (without barcodes, for the moment). I would be very grateful if you could help me understand it better.

irCLIP adapter substrate: /5Phos/AG ATC GGA AGA GCG GTT CAG AAA AAA AAA AAA /iAzideN/AA AAA AAA AAA A/3Bio/ (same as Zarnegar et al, Nat methods, 2016).

Construction schematic for generation of irCLIP DNA adapter (no need for phosphorylation step if I buy the oligo with 5’ phosphate from IDT)

Q1) I understand that the pre-adenylation step is necessary to adenylate the 5' end of the adaptor so that it can covalently bind to the 3' dephosphorylated end of the RNA fragments. IDT will custom adenylate oligonucleotides, why don't you recommend buying the sequence already pre-adenylated? This is for cost reasons. It is much more expensive to order adenylated oligos and not guaranteed yield. Whereas the 5’phosphate modification is inexpensive.

Q2) I see from your bioRxiv 2021 preprint that the introduction of enzymatic RecJ adapter removal after 3′ adapter ligation is meant to improve the efficiency of downstream steps by minimising artefacts that can be caused by adapter carry-over. However, from a chemical point of view, I am not clear which part of the adapter is removed in this step. RecJ is DNA specific from the 5’ end, so will degrade the adapter from the 5’ end. If the adapter is ligated to RNA, the 5’ end will be RNA, hence ligated products are protected.

Q3) what do you need the biotin at the end of the IR L3 adapter for? to capture and purify final cDNAs with streptavidin beads? Or for chemiluminescence RNA detection as in seCLIP 2022?

For our iiCLIP protocol, you don’t need the biotin. You can replace it with something like /3ddC/ or any other modification that will block ligation (to minimise adapter concatamers).