PAR-CLIP transition ratio is low!!!

[ckkevin911]

Hi,

I am recently working on PAR-CLIP. I used UV 365nm crosslinker from UVP company. It was irradiated twice. However, after HiSeq, the transition ratio is very very low...

Could anyone help me, please? Thank you so much!

Moreover, is there any good control for such kind of crosslinking methods?

Thank you so much for reading.

Best,

[mascano]

how much 4SU did you use for the cells?

[ckkevin911]

Thank you for your reply.

The final concentration of 4SU was 200 microM.

Doubled the concentration of original paper.

Any suggestion, please?

Thank you.

Best

[mascano]

Several things, for clarification:

Please provide more detail as to your cell culturing conditions and 4su incubation.

When you say the transition ratio is low, how are you calculating?

Are you working on a bona fide RBP (or at least using a + ctrl RBP)?
Is this RBP well-expressed and cytoplasmic? How many cells/plates?
How well did you crosslink, by post-IP stage (how much radiolabel)?

[ckkevin911]

Hi, Thanks for your reply and sorry for the late response.

I used HeLa cell. Normal 11965 media with 10% FBS and 1% pen-strep.

The incubation condition is 37 degree 16 h.

The transition ratio is calculated as: mutation event/total mapped reads

The other issues have been considered and were okay.

Thank you so much.

Best

[ckkevin911]

Hello,

Could anyone help me on this part, please?

Thank you so much.

Best