I am doing ATAC-Seq with OMNI-ATAC-Seq protocol (Kaestner Lab 2019). I did PCR amplification for 5 cycles of my library. Now I use 5ul of the PCR product to do a qPCR to determine how many additional cycles I need.
I have 4 questions about the detail of how to do this qPCR.
1. What method should I choose on the qPCR program, absolute quantification, relative quantification or delta delta CT or any other method? Should I add melt curve steps after the PCR program?
2. How much ROX did you add in your reaction system? None of the ATAC-Seq protocol (either omni-atac seq or Buenrostro's protocol) mentioned how much Rox should be added in the reaction system. We have a AppliedBiosystem QuantStudio 3 real time PCR machine which requires low rox. I usually purchase commercial SYBR GReen mastermix which includes rox, sybr green and everything in it. But in every ATAC-Seq protocol, it just said to add 0.09ul Sybr green I, it didn't mention how much Rox, or how to set up the setting of the reaction.
3. What is the amplification curve of your qPCR for ATAC-SEQ? I added low rox dye and everything else following OMNI-atac-seq protocol, but my amplification curve looks very weird. It's not a smooth curve, it's disconnected.
4. Some people mentioned to use KAPA Library Quantification kit to do this qPCR and determine number of additional cycles needed. We have the kit. Could anyone advise how to do this in detail?
Thank you!
Hi Xia,
We have done this procedure multiple times now. Regarding your questions:
1. We run qPCR with the comparative Ct template without a melt curve. The objective is to look at the linear amplification profile to determine a cycle number around 1/3 fluorscent maximum. In our hands, sometimes the 1/3rd max value is too many cycles. So, I would suggest taking a test sample and titrating different cycle numbers after the initial 5 cycles and observing the final library profile. You will see that you need a certain number of cycles to get the subnucleosomal and the mono-nucleosomal fragments at a good ratio. Typically 2:1 (approximate) would give a good profile.
2. No Rox needed as you are only looking for the amplification profile of that one sample. There's no normalization needed between replicates (we don't run replicates and running the one sample is sufficient) nor between wells. Just set up the qPCR reaction as shown with SYBR (we use EvaGreen).
3. You are looking for the linear Delta Rn vs cycle number plot.
4. Kapa kit can be used for the final libraries to determine concentration to put on the sequencer and/or for pooling. The above-mentioned qPCR is used for determining the cycle number for the final library amplification.
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