Rn/cycles: qPCR decided ATAC-seq library amplification

[zhaolin]

Hello, I have tried to set up a experiment following the ATAC-seq protocol. when I ran the qPCR side reaction to decide the cycle for library amplification, I met the problem.

The protocol uses Y-axis Rn 5000 RF as the threshold to decide the # of cycle since it is corresponded to ¼ of maximum fluorescent intensity.
In my experiment, I have tried cells # from 50,000 to 1,000,000 (the concentration of start PCR template DNA is 50 ng to 300 ng) but the maximum RF is around 200 RF(the amplification curve is fine). Should I only set 40 or 50 RF as threshold? (The correspond cycle is around 5.)
Has any one have similar issue? Could anyone help me here?

[obischof]

Hello, I have tried to set up a experiment following the ATAC-seq protocol. when I ran the qPCR side reaction to decide the cycle for library amplification, I met the problem.

The protocol uses Y-axis Rn 5000 RF as the threshold to decide the # of cycle since it is corresponded to ¼ of maximum fluorescent intensity.
In my experiment, I have tried cells # from 50,000 to 1,000,000 (the concentration of start PCR template DNA is 50 ng to 300 ng) but the maximum RF is around 200 RF(the amplification curve is fine). Should I only set 40 or 50 RF as threshold? (The correspond cycle is around 5.)
Has any one have similar issue? Could anyone help me here?

Very much depends on your PCR machine but we have similar data. On another note, if you put your amplified material on a gel or bioanalyzer do you observe a nuclesome-like pattern or rather a smear? We have trouble getting this defined nucleosomal pattern as shown in the protocol.

[zhaolin]

I haven't start doing the gel checking. But I would change the transposase digestion time if I got a smear.

[obischof]

We did; always smear no matter how long or short you incubate.

[zhaolin]

We did; always smear no matter how long or short you incubate.

What is the size of your library smear? I have tried to lower the transposase amount in the reaction and found the size of PCR product changes. But it still a piece of smear. How much of PCR product did you check on gel? Is it too low amount in the protocol?

[Wonghe]

I also got a smear for my PCR product between 150-2500bp. Has anybody sequenced these DNA does it enriched the open chomatin?

[zhengxiang he]

Hello, I have tried to set up a experiment following the ATAC-seq protocol. when I ran the qPCR side reaction to decide the cycle for library amplification, I met the problem.

The protocol uses Y-axis Rn 5000 RF as the threshold to decide the # of cycle since it is corresponded to ¼ of maximum fluorescent intensity.
In my experiment, I have tried cells # from 50,000 to 1,000,000 (the concentration of start PCR template DNA is 50 ng to 300 ng) but the maximum RF is around 200 RF(the amplification curve is fine). Should I only set 40 or 50 RF as threshold? (The correspond cycle is around 5.)
Has any one have similar issue? Could anyone help me here?

How about your experiment now?
We try several times. We start with 74k cells and can see clear cell pellet. But after we run the qPCR, we got very few signal on the qPCR and the curve is not good. Can you give us some suggestion?

[pritb]

Hello All

I am new to ATAC seq experiments. I am working on human fibroblast with 50k cells (counted through countess). Initially, I started following J Buenrostro (2015) protocol but seems not working. Now I shifted myself to Omni-ATAC protocol.

My Initial OD (after Tn5 treatment) using Nanodrop gave 46 ng/ul. I proceeded with Library prep by giving 5 cycles. Realtime PCR for library quantification gave me 20 additional cycles for my Libraries. After PCR cleanup, I got 96 ng/ul as library concentration. On gel check, I cant seen anything, except for few bands that is less than 100 bps. What could be reason?

I have attached my gel picture here.
Lane-1: 2 ul of sample after Tn5 reaction post cleanup
Lane-2: 15 ul of sample after 35 cycles (Although I selected 20 cycles based on 1/4 fluorescence intensity)
Lane-3: 10 ul of sample of 20 cycles (additional PCR cycles). This is purified. You can seen 2 bands less than 100 bps.

[Rosmano]

Those two bands with less than 100bps are adapter dimers.

I am having problems in the step of pre-amplification.
After pre-amplification I get a DNA quantity of 7.6ng/uL from 50,000 total thymocytes. However, when I try to run a qPCR I get no amplification in the 20 cycles. I ordered the TDE1 individually and directly from Illumina now that they discontinued the Nextera DNA kit, so I wasn't expecting to have problems with it.
The primers I am using are the ones from the Buenruostro paper. I'm at a loss for why I am not getting amplification in the qPCR to determine how many extra cycles I need. It seems my problem is with the primers.

What's the usual DNA concentration one has for 50,000 cells after TN5 treatment and after 5-cycle pre-amplification?

[Rosmano]

I am doing the qPCR quantification with Power SYBR Green PCR Master Mix from Thermo Fisher instead of the NEB Next HF Master Mix.

Does anyone know if this might impact the qPCR quantification?

[salix]

I am doing the qPCR quantification with Power SYBR Green PCR Master Mix from Thermo Fisher instead of the NEB Next HF Master Mix.

Does anyone know if this might impact the qPCR quantification?

Were you able to figure out what was going wrong? I am experiencing the same thing.

[Rosmano]

Yes, I needed to use the NEB Next HF Master Mix and then the quantification worked accurately.