I am trying to do ATAC-Seq from frozen brain tissue samples. Until now all protocols are from cell lines. I am looking for a good enough mechanical protocol, i.e., not using any enzymes to dissociate cells from tissue. If anyone can suggest any good protocol that would be great. I am doing bouncing tissue in cell media and strain through 70uM cell strainer, wash them with pbs, centrifuge at 300g for 5 minutes.
Why not use a solution of trypsin? When u dont want to use enzyme is the cell strainer the best
The key to obtaining a single cell suspension is to include a treatment of DNAse I to the sample. This breaks up clumpy lysed cells and frees the single cells. You can actually see the debris dissolve. You can due this alternately, Trypsin for a short time, then add DNAse I until the debris dissolve, purify the cells from the supernatant, repeat the trypsin/dnase step to the pellet to obtain more yield. I try to avoid pipetting up and down as it causes lysing (and more genomic dna which binds the cells in a gooey mess). This is the basis for the Miltenyi gentle dissociation kit
Paramita,
It seems to me that your starting point, frozen brain tissue, is far from ideal. Cellular and sub-cellular integrity is already destroyed - or at the very least greatly compromised. If the method you want to use, depends on substantially intact DNA to be able to insert Tn5, as the chromatin unravels, you are going to encounter difficulty starting with frozen tissue
It would be much better if you could procure fresh tissue. But if that is not possible, yu may be able to recovered partly sheared chromosomal DNA by using proteinase K digestion in the presence of SDS at elevated temp.
Best,
Can anyone please share me the protocol for making single cell suspension from brain tissue
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