single cell ATAC-seq and single nucleus ATAC-seq are same or different?
single cell ATAC-seq is feasible to apply on cryopreserved tissue sample or single cell suspension?
Usually, how many cells are necessary to get enough information for one sample in single cell ATAC assay?
“single cell ATAC-seq and single nucleus ATAC-seq are same or different?”
In practice, the same. A nuclei extraction from yours sample is required to remove the mitochondrial fraction of the cell. If these are not removed, they would make up a considerable proportion of your sequencing depth. When performing this single nucleus extraction, it is beneficial to consider that the buffers used are compatible with downstream applications, particular the buffer that you resuspend nuclei into prior to adding transposition enzyme mix.
This blog post is helpful and it links to a demonstrated protocol that is compatible with the 10x Genomics single ‘nucleus’ ATAC products: https://www.10xgenomics.com/blog/demonstrated-protocol-for-nuclei-isolation-for-single-cell-atac-solution
“single cell ATAC-seq is feasible to apply on cryopreserved tissue sample or single cell suspension?”
Because single nuclei are required as input to ATAC workflows, and single nuclei approaches are generally compatible with frozen tissue samples, you theoretically can use either as an input. The optimization required for nuclei extraction should not be overlooked though, and you will likely find that generating robust nuclei suspensions from solid tissue will be more work than from a sample that is already as a single cell suspension. That being said, direct nuclei extraction from solid tissue is preferable over a stepwise single cell dissociation followed by nuclei extraction. Data showing that single nucleus ATAC assays perform better when everything is done without delay are pretty convincing.
There are several protocols and even some commercial solutions for extracting nuclei. It should be noted that you would want to make sure it is compatible with the downstream assay you are looking to use.
Although this may not be specifically tested with all ATAC assays (and SCAF has not tested yet), this may be worth looking at: https://www.protocols.io/view/isolation-of-nuclei-from-frozen-tissue-for-atac-se-6t8herw
This JoVe tutorial from Dr. Ariel Levine’s lab is helpful for seeing the process of nucleus extraction in general: https://www.jove.com/t/58413/isolation-adult-spinal-cord-nuclei-for-massively-parallel-single
“Usually, how many cells are necessary to get enough information for one sample in single cell ATAC assay?”
Speaking only for the 10x Genomics single nucleus ATAC assay – other avenues will require more or less input sample .
If we start by talking about what we put into the microfluidic device for partitioning and barcoding, the simple answer is that if you want 6,000 – 7,000 snATAC-Seq datapoints you will want to have as input ~10,000 nuclei. You can target something more like 10,000 datapoints by putting in ~15,000 nuclei, but your expected doublet rate (percentage of data-points likely representing more than one cell) would be >7.5%. In terms of how many datapoints you need to have interpretable data, this is very much dependent on your biological question and factors such as the diversity of ATAC signal you are looking to interrogate. You can increase the datapoints by performing replicate captures (increasing the number of capture runs / lanes), but this can become costly. If you expect you will need extremely high throughput to generate many datapoints, consider looking at some of the combinatorial indexing strategies or more ‘home-brew’ hybrid techniques.
In terms of how many cells or how much tissue do you need to get a certain number of ‘ready-to-capture’ single nuclei. This depends on your tissue and how robust your single nuclei extraction is. Don’t be surprised if your yield varies from one preparation to the next, so it is better to plan to use a little bit more sample than the bare minimum.
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