[gpmc]

Hi,

I have been pretty consistently generating the same looking BioA traces using the FAST-ATAC protocol. Typically, double-sided SPRI removes a significant portion of the larger peak. Sequencing these libraries yields similar metrics to what has previously appeared in the literature. Unsure what that large peak is though? Perhaps it is mito-gDNA?

[Alpacino78]

Dr.

Hi there,

I am dealing with the same issue. Did you resolve this issue? I would appreciate if you share your troubleshooting experiences with me.

Thanks,
Alpa

Hi all,

[ATTACH]4740[/ATTACH]

I am new to ATAC-seq, and am testing a few different conditions based on the original protocol and a newer protocol from the greenleaf lab that was designed for blood cells (fast-ATAC). I made 4 libraries, using either 10k or 100k cells for each method. I used a 50uL reaction, using 2.5 uL of enzyme and incubating at 37C for 30 minutes. After PCR (with cycle number titration) I ran the libraries on an agarose gel and size selected the fragments between 150 and 800 bps (a post-doc in the neighboring lab has done this and has gotten good data from this).

I have attached the BA trace for 100k and 10k using the original method (IGEPAL) and the new method (fast-ATAC). The post-doc has told me that it's best not to sequence any of these because the 3n nucleosomal fraction is much higher than the sub-nucleosomal fraction (~190 bps), however I am inclined to think that the "10k fast" library would give me good data.

Does anyone have experience with taking ATAC from library prep all the way to data analysis and can comment on whether any of these four libraries is worthy of sequencing?

Thanks!