Hi, has anyone does ATAC-seq on formaldehyde fixed nuclei? I've heard it reduces the library efficiency but I have some fixed cells in the freezer and would like to get some chromatin accessibility information from them.
Hi Elsie,
Buenrostro and colleagues, have published a new protocol for ATAC on fixed cells, try to see Chen et al. 2016 the trick is to add a bit of detergent.
Link: https://www.ncbi.nlm.nih.gov/pubmed/?term=27749837
Hi Elsie,
Could you please let me know how your library preparation went with fixed cells? I have very small sample size. I am planning on fixing the cells. I am looking forward to know your experience about this technique.
Thank you,
SG
Hi Sumana,
Unfortunately I didn't end up doing ATAC-seq at all on my samples, perhaps you could contact the authors of the paper that Mads provided. Why are you fixing your cells as opposed to using the standard ATAC-seq protocol?
Good luck!
Elsie
Hi Elsie,
I am working with very less number of cells at the moment. I don't have any quicker way to get more number of cells other than fixing. That's why I would like to try fixing protocol and see if it works for my cell type.
I will contact the authors to know their suggestions regarding this.
Thank you and good luck to you as well.
SG
Hi Sumana,
For fixed cells, please add the reverse-crosslinking step (with final concentration of 50mM Tri-cl, Ph8.0, 0.2M NaCl, 1% SDS, 10ng/ul Proteinse K, 65 degree overnight, ) after the normal Tn5 tagmentatin, and then do purification---> do the PCR, all the other protocol is same.
Let me know if you need more information.
Hi Xingqi: have you performed ATAC sequencing on FFPE sections of tissues? we may have no more than 50-100,000 relevant cells available. Any helpful hints are welcome.
Unfortunately, ATAC-seq does not work well in the FFPE section tissue, the fixation is too strong there. The fixation we had introduced is same as standard ChIP-seq protocol, 1% FA fixation for 10mins (must be methanol free).
This is probably true and I believe as weel as the ATAC-seq does not work as well in the FFPE section tissue
Hi has anyone succeeded with ATACseq on FFPE or fixed nuclei? Which protocol is best?
Hi Xingqi , i'm very happy to see you here. i have two question ,the first is that in the 2013 Buenrostro' paper, you used 0.1% IGEPAL CA-630, while in 2016'paper ,you used 0.05% IGEPAL CA-630 in fixed cell, less IGEPAL CA-630? The second is that , after the PCR, have you done the size selection ? Thank you ,hope to get your words@ Xingqi Chen
Hi Elsie,
I have tried ATAC-seq on fresh frozen and 4% PFA-fixed tissues (for up to 4h). After tagmentation, we used the MinElute kit column purification. I believe the PB buffer in the kit denatures all the proteins. We got nice ATAC-seq tagmentation profiles on the bioanalyzer.
It is possible to do ATAC-seq in FFPE samples now.
see our recent manuscript below:
Profiling chromatin accessibility in formalin-fixed paraffin-embedded samples
doi: https://doi.org/10.1101/2021.07.08.450856
https://genome.cshlp.org/content/early/2021/07/14/gr.275269.121.abstract?cited-by=yes&legid=genome;gr.275269.121v1
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