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Hello,
I am attempting to perform ATAQ-seq on human macrophages. I have performed the protocol, though have encountered an issue at the qPCR step (after the initial 5 cycle amplification step). I find i have no amplification at all.
I was worried it was because i lost my cell pellet during the initial wash stages so i have nanodropped my samples at various steps:
1. After the initial purification following the transposition reaction
2. After the initial 5 cycle PCR amplification step
3. After final purification step
However, i have DNA at all of these steps, indicating i haven't lost my pellet and the DNA is being amplified.
For this protocol i am using the Illumina Nextera Index Kit (FC-121-1012) as the primers.
Regarding this i have two questions:
1. Are these indices compatible with qPCR?
2. For each sample should use the same index pair used in the amplification PCR in the qPCR?
Any other suggestions would be greatly appreciated!
Thanks
I am attempting to perform ATAQ-seq on human macrophages. I have performed the protocol, though have encountered an issue at the qPCR step (after the initial 5 cycle amplification step). I find i have no amplification at all.
I was worried it was because i lost my cell pellet during the initial wash stages so i have nanodropped my samples at various steps:
1. After the initial purification following the transposition reaction
2. After the initial 5 cycle PCR amplification step
3. After final purification step
However, i have DNA at all of these steps, indicating i haven't lost my pellet and the DNA is being amplified.
For this protocol i am using the Illumina Nextera Index Kit (FC-121-1012) as the primers.
Regarding this i have two questions:
1. Are these indices compatible with qPCR?
2. For each sample should use the same index pair used in the amplification PCR in the qPCR?
Any other suggestions would be greatly appreciated!
Thanks