HI all
I am trying to produce ATAC-seq libraries from transfected cells, but it seems a pain...
I have produced good libraries from non-transfected cells (sequencing was OK), but when I transfect cells with a plasmid I get, of course, a lot of tagmentation in the plasmid, such that most of my reads are plasmid-derived. I would like not to diminish the plasmid concentration, because all my experiment so far have been done using the same conditions.
We are trying to solve this problem but it does not look easy...
My idea is to get rid of the plasmid before tagmentation and probably use AMPure beads to suck it up just after lysing the cells. But I am not sure how this will work because there may be complications (I am going to test this next week, I think)
Another possibility is to check the tagmented genome (before amplification) on a gel and see if it is be possible to separate the plasmid from the genome. This actually raises the question if the genomic DNA is fragmented after tagmentation (I think it is, unfortunately... can you confirm?)
We were also considering sequence capture to get rid of the plasmid fragments, but this also has some problem in the fact that the adapters may then hybridise together resulting in non specific sequence capture.
Please let me know if you have found such a problem and came up with a solution.
Very much appreciated
Bests
Dam
I am trying to produce ATAC-seq libraries from transfected cells, but it seems a pain...
I have produced good libraries from non-transfected cells (sequencing was OK), but when I transfect cells with a plasmid I get, of course, a lot of tagmentation in the plasmid, such that most of my reads are plasmid-derived. I would like not to diminish the plasmid concentration, because all my experiment so far have been done using the same conditions.
We are trying to solve this problem but it does not look easy...
My idea is to get rid of the plasmid before tagmentation and probably use AMPure beads to suck it up just after lysing the cells. But I am not sure how this will work because there may be complications (I am going to test this next week, I think)
Another possibility is to check the tagmented genome (before amplification) on a gel and see if it is be possible to separate the plasmid from the genome. This actually raises the question if the genomic DNA is fragmented after tagmentation (I think it is, unfortunately... can you confirm?)
We were also considering sequence capture to get rid of the plasmid fragments, but this also has some problem in the fact that the adapters may then hybridise together resulting in non specific sequence capture.
Please let me know if you have found such a problem and came up with a solution.
Very much appreciated
Bests
Dam
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