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Old 04-20-2017, 07:07 AM   #1
Vgamma4
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Location: Europe

Join Date: May 2016
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Default ATAC-seq small / overdigested fragments?

Hi all,

First, I hope that the ATAC-Family will grow further to share their experiences here.
My experience with ATAC-sequence is kind of confuse. At the beginning I FACS sorted cells (20k - 50k) and followed the original Greenleaf paper. The fragments measured by BA were very small (one peak at 180bp). See Pic1

Then I simplified the process by just taking freshly isolated cells from lymph nodes, tried different cell numbers from (10k - 100k), with and without lysis (as decried in other threads. Suddenly I got this typically nucleosome pattern and run those samples at the HiSeq (see Pic2). The results were promising. mtDNA reads were without lysis at 10% and with lysis around 40%. 25k cells seemed also optimal for sequencing.

Then I went back with my optimised setting to the FACS sorting cells and got again only small fragments peak at 180bp. The sort by itself looked good.

Could it be that sorting cells influences the quality of the libraries meaning that the cells are stressed or maybe already dying. Any experience with small fragments? Could it be that the DNA is overdigested?

I am happy for any suggestions. Thanks
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Old 08-30-2017, 06:41 PM   #2
mkel93
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Location: Melbourne, Australia

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Hello,
Did you ever optimise your protocol for sorted cells? I am having the exact same problem trying to do ATAC on 50K sorted haematopoietic cells (KSL).
Thanks
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