Hi everyone. I am a new one in ChIP-seq and planning to do it for a poorly characterized TF. We are going to do ChIP on transfected HEK293 cells with WT and 2 other mutant forms of our specific gene which makes three samples to be sequenced. Regarding the negative control "Input DNA", the amount of samples for NGS would be 6 samples (1 sample+ its corresponding input DNA) which is quite expensive for us. As I am searching to find a way to reduce the cost, I was wondering that if its possible to find the sequencing RAW files of control DNA for HEK cell somewhere in databases and use it as a control to normalize the analysis. Is it possible? Are there such kind of data available and if so, can we use them instead of input DNA? As I understood the sonication is not a truly random process and we may have to sequence each samples's input DNA separately.Am I correct? If so, can we sequence only one of the input DNAs (for example WT) as the control for all the three samples or we have to consider one seperate input DNA for each sample?
I am looking forward to hearing from you.
Typically, you take a sample of sonicated chromatin, split it in to X tubes, where X is the number of antibodies to try +1, then the +1 goes through all the same library prep stages except it was never bound to magnetic beads and washed. That is input. So you don't have an input per antibody! That would be quite expensive :P
Dear John, Thanks for your reply. Actually, the problem is that I am going to sonicate three similar HEK293 cell lines which are all transfected with a same gene but in WT and mutant forms and for all of these I only use one antibody (anti-GFP). Can I still use one "input DNA'' for all 3 samples (3 different sonicated chromatins)?
I see. hm. Well this isn't really a bioinformatics question, so i'm afraid the answer might leave a bitter taste in the mouth of my peers, but essentially the answer to your question depends on how "exploratory" this experiment is. The correct answer is "yes, every single sample of the 6 needs an Input control", since the input chromatin is different every time and you don't have biological or technical replicates. However, practically, this could be a waste of time and money..
I'm guessing you're using GFP because you previously did imaging work, found that it localizes in the nucleus, found that it also binds DNA, and now you want to see where it goes but you don't have the time/money to generate a ChIP antibody for the actual protein? (this is under the assumption that the TF was really poorly characterized as per your OP). This is already puts you into the realm of "not conclusive" if you wanted to publish this data, since sticking a huge molecule like GFP onto the protein changes how/where it can bind DNA. Furthermore, GFP-ChIP is notorious for having high background noise (if you tag the protein with something for GFP to bind to, rather than make a hybrid protein). Far better to spend money on buying/generating the antibody than the 6 Inputs. Or alternatively use some new commercial thing that will overcome the issues with the GFP. This would be a better use of your money than Input controls.
Finally, and i feel a bit naughty saying this, but you can always generate a ChIP'd library and an Input library from each of the 6 samples, then not sequence the Inputs unless it becomes necessary. Libraries can be stored for many many years, and an input costs essentially nothing other than your time to generate. You can pull it out of the freezer and sequence it if the reviewers ask for it. Really though, your WT sample is a better control than input anyway. At least, for an exploratory analysis for the effect of the mutation, and so long as you do the fixation and sonication on samples processed all on the same day with the same buffers, etc. Don't be using different fixatives or fixation durations. That's when your input control is most valuable. Don't be sonicating Line 1 on Monday, then Line 2 on Friday after Dave from Accounting has been fiddling with the machines power output to save a few cents. Keep it nice and consistent, and maybe, just maybe, you'll probably have to sequence the Input anyway ;-)