Different PCR signal from input control after library prep

[AveragePhD]

Hi all,

I used the Bioo Scientific kit to make my barcoded ChIP-Seq libraries. The signal by PCR was similar across all 3 input control samples under different conditions. However, they became so different after I made the libraries. Strangely, my sample libraries have maintained specific enrichment of target sites. The bioanalyzer results look good. It seems to be a selection during the library prep? Has anyone encountered this problem?

Any suggestions are welcome!

Thank you in advance!

[pmiguel]

"so different"?

Show us the images of the differences before/after library construction.


--
Phillip

[AveragePhD]

Hi Phillip,

Please see the attached gel image.

Thanks.

[pmiguel]

Okay, I see.
Not ideal, but they might be okay. How many cycles of enrichment PCR did you do after ligating on the adapters?
Did you do qPCR to determine your yield?
--
Phillip

[AveragePhD]

I did 4 cycles of PCR before size selection on the gel, followed by 14 cycles of PCR to enrich the library.

No, I didn't do qPCR because we can't afford SyBr gren.

[AveragePhD]

Is it ok to do different cycles to enrich the library across different samples even they are to be compared? Should I use more cycles for sample 1 and less for 2 to make it more even??

[pmiguel]

Sure. If you take a look at Ethanol's blog, you will see that is part of his normal protocol.

--
Phillip

[AveragePhD]

Thanks Phillip!

[AveragePhD]

Smaller pcr product than expected in ChIP-seq library enrichment confirmation

Dear Phillip,

Thank you so much for your suggestions last time and the ChIP-Seq libraries worked well. I just prepared more libraries and when I checked the enrichment of target sites, one of my sample had a smaller pcr product than expected (please see the attached pic)!!! What do you think might have caused this?

Thank you in advance!

[AveragePhD]

Any inputs from anyone is welcome!!

I couldn't start a new thread for some reason.

Thank you all.

[pmiguel]

Dear Phillip,

Thank you so much for your suggestions last time and the ChIP-Seq libraries worked well. I just prepared more libraries and when I checked the enrichment of target sites, one of my sample had a smaller pcr product than expected (please see the attached pic)!!! What do you think might have caused this?

Thank you in advance!

Did you do a size selection (gel cut, for example) at some point? Your bands are really tight, so my guess is that gel cut hit a different size than the others. (I notice your gel appears to have a substantial frown. Maybe during the gel cut you were excising straight across lane-to-lane but size profile varies lane to lane.)

Probably want to ask someone that does a lot of ChIPseq.

--
Phillip

[AveragePhD]

Hi Phillip,

Yes, I did size select the inserts and I agree that there was some variation between the gel slices but I don't understand how the size of the pcr product got smaller using the same primers.

[ETHANol]

What are the size markers. Is the band in lane 1 about 120 bp. If so it is adapter dimer? Which would lead to the question of why the samples in lanes 2,3 and 4 are so small.

[AveragePhD]

Hi Ethan,

This was a 100 bp marker. Just to clarify, the gel shown is the pcr reaction to check the enrichment of my target site NOT for size selection during the library prep. So the band in lane 2 is NOT adapter primers because I used the primers of my target site to check if the libraries have preserved the same enrichment. The PCR product of my target site is about 160bp which is correct for lanes 3 and 4. I just don't know why I got a smaller product in lane 2.

Thank you for your reply.

[AveragePhD]

By the way, I used the Bioo scientific kit to prepare the ChIP-Seq libraries and I cut out the gel slice around 300 to 500 bp which should not contain adapter dimers.