Q1: What is the sensitivity of the enzyme to different cell types?
Once the nuclei are isolated, the enzyme sensitivity should be similar between different cell types. However, some cell lines will be more sensitive to the detergent in the Nuclei Prep Buffer and thus result in a loss of nuclei. You can dilute the Nuclei Prep Buffer 1:1 with dsDNase Digestion Buffer prior to nuclei isolation.
Q2: What is the difference between Atlantis dsDNase and MNase?
• Atlantis dsDNase is a double-stranded DNA specific endonuclease that cleaves phosphodiester bonds in DNA to yield oligonucleotides with 5’-phosphate and 3’-hydroxyl termini. Increasing the incubation time with Atlantis dsDNAse will increase efficiency and generate more mono-nucleosomal DNA.
• Micrococcal Nuclease (MNase) is a single and double-stranded DNA and RNA endonuclease. MNase is more specific for single-stranded nucleic acids, but cleavage is biased towards AT-rich and AU-rich sequences. MNase will result in more robust digestion compared to Atlantis dsDNase. Increasing the incubation time with MNase will increase efficiency of this enzyme and generate more mono-nucleosomal DNA.
• The enzyme selection depends on desired downstream applications.
Q3: Do the sticky-end fragments have 5’ vs. 3’-overhangs? What is the relative abundance of different ends?
Atlantis dsDNAse generates random-end, i.e. both blunt- and sticky-end DNA fragments and there is no specific length for overhangs.
Q4: What if the nucleosomal DNA is not shearing properly and the majority of DNA is still intact?
Washing the trypsinized cell pellets with PBS is very critical step. Residual EDTA in the cell pellets will decrease both Atlantis dsDNAse and MNase digestion efficiency.
Other considerations for incomplete shearing:
• Check that the correct number of cells was digested. (D5220 kit: 1×106 mammalian cells)
• If you only want mono-nucleosomal DNA instead of nucleosomal ladder containing mono-, di-, tri-nucleosomal, etc., you will need to increase the enzyme concentration. Based on our experience, we need to use >1U per Atlantis dsDNAse per million cells.
