I have a CLIP-Seq dataset I'm processing, which includes control samples and no inputs. This is the second CLIP analysis I've performed to help out users of our genome core facility and the first one only had inputs. I've done a number of ChIP analyses in the past, which I loosely consider to be a similar type of analysis, and they all used inputs, so I've never had to look into the differences in using inputs versus controls when doing peak calling before. Now that I'm trying to figure out what I need to do, I'm a little lost.
Here's what I've got:
- Samples of cross-linked RNA/protein complex that went through a poly-A and then a FLAG pull-down (protein G beads with an antibody). The complex was eluted, treated with proteinase K, and the RNA was sequenced.
- Controls went through the same processing as the samples except the control beads had no antibody
There are replicates in both cases. (As a side-note, the RNA was treated with RNase III while bound to the beads and the RNA's trimmed off the complexes were also sequenced. I'm not sure what to do with those samples yet.)
I was going to use one of the CLIP peak callers available on Galaxy, but whichever one I use, all the protocols I've found and the documentation I've looked at use inputs for the peak calling method and not controls. So what do I need to do differently (if anything) to use the controls as the background in the peak calling? Or do I need to suggest they also sequence the inputs?
And just so I know (dumb question here)... Is "input" (as the name suggests) the sample as it was before the FLAG pulldown? Why would one use one or the other (input versus control).