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Old 11-13-2014, 11:52 AM   #1
zhaolin
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Default Rn/cycles: qPCR decided ATAC-seq library amplification

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 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?
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Old 11-13-2014, 01:19 PM   #2
obischof
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Quote:
Originally Posted by zhaolin View Post
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 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.
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Old 11-14-2014, 07:09 AM   #3
zhaolin
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I haven't start doing the gel checking. But I would change the transposase digestion time if I got a smear.
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Old 11-14-2014, 07:16 AM   #4
obischof
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We did; always smear no matter how long or short you incubate.
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Old 11-17-2014, 09:00 AM   #5
zhaolin
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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?
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Old 11-24-2014, 12:19 AM   #6
Wonghe
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I also got a smear for my PCR product between 150-2500bp. Has anybody sequenced these DNA does it enriched the open chomatin?
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Old 01-28-2015, 01:23 PM   #7
zhengxiang he
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Quote:
Originally Posted by zhaolin View Post
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 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?
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Old 03-13-2019, 01:12 AM   #8
pritb
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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.
Attached Images
File Type: jpg Presentation1.jpg (37.5 KB, 4 views)
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