Hello everyone!
I’m currently working on constructing a library for ATAC-seq with mouse intestinal tissue. I’ve prepared a 10μm thick section of mouse colon tissue, mounted it on glass, and proceed with ATAC labeling. Unfortunately, the results are not what I had hoped for.
I’m at a loss as to what could be causing these unexpected results. Has anyone here encountered similar challenges in their work? I’m eager to hear from those with experience in this area.
To give you some context, I’ve done some calculations to estimate the amount of DNA I’m working with. Assuming the colon has a diameter of 2500μm and a lumen radius of 1000μm, and considering the section thickness is 10μm with a cell diameter of 10μm, I estimate there to be approximately 164,850 cells in the section. With Thermo’s data suggesting each cell contains 15 pg of DNA, the total for my section would be around 2,472.75 ng. If only 5% of this DNA is accessible chromatin (AC) DNA, that would amount to roughly 123.64 ng.
Based on these calculations, I selected a kit that suggests a 50 ng DNA input. To account for the estimated AC DNA in my sample, I opted to use 4 times the standard amount of enzyme recommended by the kit for a single section of the intestinal tissue. I’m looking for feedback on whether this approach is sound, or if there might be any drawbacks to this method.
Attached is the electropherogram of my library, which was obtained from the Agilent 5200. As you can see, it’s not up to the standard I was aiming for.
I’m currently working on constructing a library for ATAC-seq with mouse intestinal tissue. I’ve prepared a 10μm thick section of mouse colon tissue, mounted it on glass, and proceed with ATAC labeling. Unfortunately, the results are not what I had hoped for.
I’m at a loss as to what could be causing these unexpected results. Has anyone here encountered similar challenges in their work? I’m eager to hear from those with experience in this area.
To give you some context, I’ve done some calculations to estimate the amount of DNA I’m working with. Assuming the colon has a diameter of 2500μm and a lumen radius of 1000μm, and considering the section thickness is 10μm with a cell diameter of 10μm, I estimate there to be approximately 164,850 cells in the section. With Thermo’s data suggesting each cell contains 15 pg of DNA, the total for my section would be around 2,472.75 ng. If only 5% of this DNA is accessible chromatin (AC) DNA, that would amount to roughly 123.64 ng.
Based on these calculations, I selected a kit that suggests a 50 ng DNA input. To account for the estimated AC DNA in my sample, I opted to use 4 times the standard amount of enzyme recommended by the kit for a single section of the intestinal tissue. I’m looking for feedback on whether this approach is sound, or if there might be any drawbacks to this method.
Attached is the electropherogram of my library, which was obtained from the Agilent 5200. As you can see, it’s not up to the standard I was aiming for.