Multiplexing RNA-Seq and ChIP-Seq in same lane

[menzies]

Is it OK to multiplex RNA-Seq and ChIP-Seq samples into the same lane? My ChIP is sheared to around 300bp using sonication, and the RNA-Seq samples are fragmented to 300-400bp by cationic RNA fragmentation.

The only problem I can think of is the effect of different-sized fragments on qPCR library estimation (before mixing to multiplex the samples). Are there any other problems I haven't thought of?

[UKboston]

I just asked Illumina tech support about this:
In general, multiplexing ChIP-Seq libraries is possible but not supported since it is difficult due to low input material levels, high number of PCR cycles necessary, and inefficiency of the three-primer multiplex PCR. For TruSeq Cluster Kits it is not possible to do multiplexing of ChIP-Seq with Small RNA v1.0, v1.5. Please find this informaiton also on website: http://support.illumina.com/sequenci...atibility.ilmn

[luckylove]

I just asked Illumina tech support about this:
In general, multiplexing ChIP-Seq libraries is possible but not supported since it is difficult due to low input material levels, high number of PCR cycles necessary, and inefficiency of the three-primer multiplex PCR. For TruSeq Cluster Kits it is not possible to do multiplexing of ChIP-Seq with Small RNA v1.0, v1.5. Please find this informaiton also on website: http://support.illumina.com/sequenci...atibility.ilmn

do you mean that chip-seq samples should be sequencing alone in one lane. and in this lane the best thing is there is no other kind of sequencing samples? thank you!

[UKboston]

That's what it sounds like to me, though the reasons are unclear to me. I read elsewhere that, while not recommended, it is possible to multiplex ChIP-seq libraries. I haven't done it and have no immediate plans to do so.