[smallcreek]

Hi guys,
I am happy to read your valuable discussion here. We recently had problem that the color balance of the sequencing is completely off, which caused majority of image pannel failed. On the WFA, every parameter looks fine except P2_Gain is too low (lower than 8). Is that color balance off is caused by the concatamerization of adaptors? We did not know this trick and did not dilute the adaptors while our ChIP DNA concentration is very low. If the P2_gain is a result of adaptor concatamerization, we can use it to titrate the adaptor dilution based on the concentration of ChIP Seq samples from our customers.

Thanks in advance

Thanks in advance.

Smallcreek

[rmetz]

Did you by chance use salmon sperm or another DNA as a block or carrier during the chIP?

(We've had 2 separate groups who did just this. Leads to very low % alignment. Uggg! On the other hand were up to 100million salmon reads if anyone wants to take a go at a denovo assembly.)

Has anyone else had this problem?

[Hamid]

Hi Elly,

Shearing to a specific size range does not necessarily correspond to efficient IP. Often people expose their chromatin to too much energy which provides great shearing results at the expense of epitope integrity. I would suggest that you carry out a western of your sheared material with the same antibody you are using for your IP. It is a cheaper and an effective method of QC for ChIP. We routinely do that in our lab, and the results correlates very well with our IP. You can also carry out qPCR with epitope specific primers.

Thank you

Hamid

[captainentropy]

Did you (elly) run your libraries on a bionalayzer? If your library is mostly amplified adapters then what you observe is exactly what you would expect. A bioanalyzer trace would very clearly indicate this with a strong peak in the 124bp range.

And if you have access to an instrument like a Pippin Prep or a LabChip XT then you should use that. It will pretty much eliminate adapter contamination prior to the PCR step (assuming you aren't size selecting below say 150bp).