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Old 03-14-2017, 07:45 AM   #1
halllb
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Unhappy Unable to get ATAC-seq to work

good morning. for the past several days, i have been attempting to perform ATAC-seq on some Jurkat cell pellets. i am following the Buenrostro/Greenleaf protocol published in Current Protocols (2016). to-date, i cannot get the tagmentation reaction to work. i have tried several different conditions; e.g., 1) running a titration experiment using 7.4K, 14.8K, 29.5K, 44.3K, 59K, and 88.5K live cells from a cell pellet assuming 59% live cells; 2) eliminating the lysis step, which i read in one of the threads on this site, using 14.8K, 44.3K, and 59K live cells. each time, i've isolated the nuclei and immediately performed the tagmentation reaction. after cleaning up the reaction, the BA traces show only one large (10,380bp) peak, indicating no fragmentation and hence, no tagging. to test the enzyme, i used commercial DNA. the tagmentation reaction didn't work, so i called Illumina. they suggested running the DNA through a column since some of the commercial gDNA preps contain EDTA to which the transposase is very sensitive. once i cleaned up the gDNA, the fragmentation reaction worked well. i was going to put the isolated nuclei over a clean-up column, but my boss informed me that it would relax most of the supercoiled DNA, resulting in fragmentation of all the gDNA, instead of just the accessible chromatin sites. does anyone have any suggestions that i can try? i have read on this site that many of you are getting very good results using the Greenfield protocol. i welcome any advice you can offer. thanks so much for your time. laura hall
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Old 03-14-2017, 08:52 AM   #2
NickPantelireis
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I read somewhere in one of the other posts on here that after the tagmentation you still need to amplify your DNA using PCR if you haven't already. The amount of DNA after tagmentation is quite a low amount so might not be being detected on the BA. The 10,380bp peak might just be the upper marker of the HS DNA assay.
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Old 03-14-2017, 09:26 AM   #3
halllb
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hi nick: thanks for replying. I also read that. I'm not sure the large peak is marker, because it's not a sharp peak...it's a rounded one. also, I can see heavy bands that look like DNA in the gel image. below is a link to a pic of the BA... what do you think?
laura
http://imgur.com/a/LCU0T
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Old 03-15-2017, 02:17 AM   #4
NickPantelireis
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Ah yeah, I see what you mean. It does look like the DNA is underdigested or as you said not digested at all. With PCR you should be able to see if your primers attach to your DNA. If not then the enzyme is definatly not tagmenting them as the primers can't attach to their complementary sequence. Otherwise, are you washing, resuspending cells, incubating @37C during the reaction?
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Old 03-15-2017, 11:26 AM   #5
halllb
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hi nick:

thanks for the reply.

regarding doing PCR, i haven't run these current samples through PCR. on a previous attempt, however, i ran the entire protocol, all the way through sequencing, with some samples that appeared to work. unfortunately, upon sequencing, we discovered that we were getting over-tagmentation and essentially performing whole-genome sequencing. a troubleshooting section in the Current Protocols chapter suggests that tagmentation needs to be optimized to cell number. hence, the current experiments with titrating cell numbers; but no fragmentation.

regarding washing, resuspending the cells, and incubating @37C during the reaction, yes i have performed all these steps with strict adherance to the protocol.

i appreciate your suggestions and questions, and look forward to hearing any additional suggestions you may have.

thanks,
laura
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