Can I increase UV intensity like 600 mJ/cm2 or multiple times (5 or 6 times using 400 mJ/cm2 setting)?

Increases in efficiency with crosslinking longer than recommended in the protocol can be seen, depending on the protein studied. However, we advise to follow the protocol described in Ule et al, Methods 2005, with 150 mL/cm2 setting for monolayer cells. Prolonged cross-linking could initiate a DNA-damage response in the cells, and can lead to crosslinking of multiple proteins to nearby sites on RNAs. This could decrease the resolution of the method, and cause co-purification of non-specific proteins and other non-specific RNAs that crosslink to these proteins.

Can frozen tissue be effectively cross-linked?

Yes, the frozen tissue pieces can be ground with mortar and pestle on liquid nitrogen (see and then the frozen powder crosslinked. This is helpful especially for tissues that are not soft enough to dissociate using the standard pipetting trituration method described in our protocol. Alternatively, if the frozen tissue is soft enough for trituration after thawing, it can be crosslinked after thawing and dissociation as described in (Ule et al., 2005).

To increase the UV crosslinking efficiency, I wonder if I can first homogenize the tissue with Dounce homogenizer, then do UV crosslinking with the supernatant after spinning, if the homogenization will not affect the complex.

Homogenisation will certainly affect the protein-RNA interactions, and could lead to non-physiologic interactions with RNAs that normally don’t co-localise with the complex in cells. I don’t think that the potential increase in cross-link efficiency is worth taking the risk of getting non-informative data.

What is the exact nature of the covalent bonds between the UV-crosslinked protein and RNA?

I assume you are referring to the biophysics behind the bond formation? I find the explanation from this paper quite nice (Erika C Urdanetabenedikt, 2020).