Hi everyone,
I've done my first ATAC-seq library preparation recently and had a few questions about the result. I would be very grateful if anyone could kindly give me some feedback/suggestions.
I used 50,000 HEK cells (fresh cells), and followed Buenrostro JD, et al. 2015 protocol exactly. After nuclei preparation -> Tn5 tagmentation (37C, 30min, on a heating block) -> PCR amplification (total cycle number was 11), I purified library with QIAGEN MinElute PCR Purification Kit and eluted in 20ul elution buffer provided by the kit. I loaded 10ul, 5ul, and 2.5ul samples on a 2% agarose gel (containing GelRed) to visualize the bands. Please see attached image.
I can see a nucleosome-like pattern at expected sizes, but not very strong. I'm wondering:
1. Is this result good enough for sequencing? Should I further optimize the condition (e.g. titrate Tn5 enzyme)?
2. There is a strong band below 100 bp (the lowest one), but I have no idea what it is. Could it be primer dimers? I have purified the library before running the gel, so I didn't expect to see primer dimers...
3. I expected to see strong signals below 150 bp, which come from sub-nucleosomal DNAs. However, I hardly see anything between 150 bp and the lowest band (indicated by a red square bracket in the image), even no background smear. Does anyone have similar experience and know why?
Thank you so much! Any comments would be highly appreciated!
Best,
Dan
I've done my first ATAC-seq library preparation recently and had a few questions about the result. I would be very grateful if anyone could kindly give me some feedback/suggestions.
I used 50,000 HEK cells (fresh cells), and followed Buenrostro JD, et al. 2015 protocol exactly. After nuclei preparation -> Tn5 tagmentation (37C, 30min, on a heating block) -> PCR amplification (total cycle number was 11), I purified library with QIAGEN MinElute PCR Purification Kit and eluted in 20ul elution buffer provided by the kit. I loaded 10ul, 5ul, and 2.5ul samples on a 2% agarose gel (containing GelRed) to visualize the bands. Please see attached image.
I can see a nucleosome-like pattern at expected sizes, but not very strong. I'm wondering:
1. Is this result good enough for sequencing? Should I further optimize the condition (e.g. titrate Tn5 enzyme)?
2. There is a strong band below 100 bp (the lowest one), but I have no idea what it is. Could it be primer dimers? I have purified the library before running the gel, so I didn't expect to see primer dimers...
3. I expected to see strong signals below 150 bp, which come from sub-nucleosomal DNAs. However, I hardly see anything between 150 bp and the lowest band (indicated by a red square bracket in the image), even no background smear. Does anyone have similar experience and know why?
Thank you so much! Any comments would be highly appreciated!
Best,
Dan
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