I received C. elegans nuclei (20 million nuclei) for ATAC-seq and prepared the library based on the Buenrostro 2015 protocol. The final cycle number was determined by 1/3rd maximal qPCR fluorescence and total cycle number used was 14. Could this be due to too high input or have others seen anything similar due to a different reason? We are going to try lowering the input significantly and titrate input amount to find the nucleosome pattern. Any input would be appreciated. Thank you.
Hello Pabalu Karunadharma, so it seems to me that your input is extremely high for this assay. If I'm interpreting this correctly, you used all 20 million nuclei in a single sample? You need to optimise it for your specific experiments, but I would not recommend using higher than 50K cells.
In our lab, we have used the protocol with 20K cells (transposition reaction mix was halved to 20ul) as well and our libraries/ sequencing profiles have turned out well. I would suggest titration of 2K, 5K, and 10K to start with. The chip looks overloaded, considering the input and the 14 PCR cycles that were run.
Hope this helps.
Thank you Chinchu! I'm sorry I didn't check on this until now. These are C. elegans cells and the lab who provided the nuclei had seen other groups use a higher number of cells. However, I agree that 20 million was too high. We did another test with titrating 0.5, 1 and 2 million nuclei. 1.5 million nuclei is approximately closer to 50K human cells as the C.elegans genome is 30-fold smaller. However, I'm still getting a smear. I think we need to optimize the nuclei prep. Thanks!
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