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Hello, I am a new user who has tried searching for an answer to a problem I am having with Atac-Seq library prep.
Let me start off by saying I am using the Buenrostro 2013 Nature Protocols paper and using 50,000 FACS purified mouse cells.
The issue I am having is with the qPCR side reaction requiring MANY more cycles than reported, with others suggesting ~10-15 addition cycles, but my samples requiring ~35-40 extra cycles!
I have attached two images:
(1) Amplification plot showing that I need to add ~37 addition cycles to get 1/3 of the peak fluorescence. You will also notice that the first 30 cycles show almost no amplification and then the reaction takes off -- not sure what this means -- potentially very low starting material? I will note that I decided for these samples to run the qPCR using a 2X SYBR Green Master Mix that contains its own Taq (Thermo Power SYBR Green Master Mix, Cat#4368577) that my lab already had as opposed to the SYBR Green I alone, but otherwise followed everything from the Buenrostro manuscript. I did this because someone else at my institute previous did the same and their side qPCR reactions worked fine and only required ~10 extra cycles. I realize now that this may have been a mistake?
(2) Gel of my amplified final library preps after all PCRs showing that I get a decent smear as reported by some, but don't necessarily have the distinct ~150bp cutoff (sorry for ripping the gel). I end up cutting out 100-1,000bp fragment and gel purifying, followed by PCR purification column, QC, and eventually Seq.
I have yet to send these samples to seq as they were my test samples before I run my more precious samples. I guess the main question I have is: If this was your experiment would you bother sending them to Seq or does taking this many cycles ruin the experiment?
I appreciate any advice the community can offer!
Thanks,
-J
Let me start off by saying I am using the Buenrostro 2013 Nature Protocols paper and using 50,000 FACS purified mouse cells.
The issue I am having is with the qPCR side reaction requiring MANY more cycles than reported, with others suggesting ~10-15 addition cycles, but my samples requiring ~35-40 extra cycles!
I have attached two images:
(1) Amplification plot showing that I need to add ~37 addition cycles to get 1/3 of the peak fluorescence. You will also notice that the first 30 cycles show almost no amplification and then the reaction takes off -- not sure what this means -- potentially very low starting material? I will note that I decided for these samples to run the qPCR using a 2X SYBR Green Master Mix that contains its own Taq (Thermo Power SYBR Green Master Mix, Cat#4368577) that my lab already had as opposed to the SYBR Green I alone, but otherwise followed everything from the Buenrostro manuscript. I did this because someone else at my institute previous did the same and their side qPCR reactions worked fine and only required ~10 extra cycles. I realize now that this may have been a mistake?
(2) Gel of my amplified final library preps after all PCRs showing that I get a decent smear as reported by some, but don't necessarily have the distinct ~150bp cutoff (sorry for ripping the gel). I end up cutting out 100-1,000bp fragment and gel purifying, followed by PCR purification column, QC, and eventually Seq.
I have yet to send these samples to seq as they were my test samples before I run my more precious samples. I guess the main question I have is: If this was your experiment would you bother sending them to Seq or does taking this many cycles ruin the experiment?
I appreciate any advice the community can offer!
Thanks,
-J