ATAC-Seq large fragments

[cfuttner]

Hi All,
We've been trying to optimize ATAC-Seq on sorted embryonic cell populations and have run into a possible problem. After library prep we QC with a BioAnalyzer and see a large peak at around 2000bp. This doesn't change with increased amounts of enzyme or increased incubation. Any thoughts?

[rbd]

Hi All,
We've been trying to optimize ATAC-Seq on sorted embryonic cell populations and have run into a possible problem. After library prep we QC with a BioAnalyzer and see a large peak at around 2000bp. This doesn't change with increased amounts of enzyme or increased incubation. Any thoughts?

I have been using ES cells and for a while I was getting profiles like that. I started getting a greater proportion of small fragment peaks when I lowered the reaction volume to 5ul and used 2.5 ul of transposase and 2.5 ul TD. You will have to resuspend your lysed cells in the tagmentation mix. Skip the cleanup step after tagmentation. This method came from the original ATACseq paper for lower cell numbers. Hope that helps.

[Tal Golan]

ATAC-seq and bioanalyzer

Hi,
I have a 'side question'- In which Bioanalyzer (and kit) did you use? I suppose you used a kit suitable for DNA library with long fragments (>1Kb)..
Thanks

[rbd]

Hi,
I have a 'side question'- In which Bioanalyzer (and kit) did you use? I suppose you used a kit suitable for DNA library with long fragments (>1Kb)..
Thanks

Its the Agilent 2100 Bioanalyzer using the High Sensitivity DNA kit. Its intended for fragments under 1KB but the upper marker is at 10KB so you can see some of the larger fragments as well.

[tubear]

how many cells you used in the reaction?

I have been using ES cells and for a while I was getting profiles like that. I started getting a greater proportion of small fragment peaks when I lowered the reaction volume to 5ul and used 2.5 ul of transposase and 2.5 ul TD. You will have to resuspend your lysed cells in the tagmentation mix. Skip the cleanup step after tagmentation. This method came from the original ATACseq paper for lower cell numbers. Hope that helps.

[Bertha77]

Perhaphs increasing tagmentation time would help you.... also make sure the cell counts are correct