How to pool ATAC-seq libraries in equimolar concentrations?

[ECorrales]

Hi! I have 24 ATAC libraries prepared for sequencing. For this, I want to pool them all together. I ran them on the TapeStation and I have the distinctive nucleosomal banding pattern (see attached files). I'm hesitating a bit on how to properly estimate the molarity of every library when I have multiple bands, since the Tape is not always detecting the same bands. Any suggestion about this? Should I just use Qubit quantification for pooling?

[kmcarr]

Originally Posted by ECorrales

Hi! I have 24 ATAC libraries prepared for sequencing. For this, I want to pool them all together. I ran them on the TapeStation and I have the distinctive nucleosomal banding pattern (see attached files). I'm hesitating a bit on how to properly estimate the molarity of every library when I have multiple bands, since the Tape is not always detecting the same bands. Any suggestion about this? Should I just use Qubit quantification for pooling?

Use Region Analysis on the TapeStation software instead of Peak Analysis to determine an average size for each sample. Set up a Region from ~150-1000bp on all samples and use that calculate the average size. Use this average size and Qubit measurements for each to then calculate a molar concentration for each library.

Before you do this though, correct the Upper Marker in wells E1, A2, C2, F2 and G2 in the 10-24 file. The Upper Marker should be manually set tot he taller of the two peaks (Upper Marker peak height should alway be almost 2X the Lower Marker peak height.)

[ECorrales]

Originally Posted by kmcarr

Before you do this though, correct the Upper Marker in wells E1, A2, C2, F2 and G2 in the 10-24 file. The Upper Marker should be manually set tot he taller of the two peaks (Upper Marker peak height should alway be almost 2X the Lower Marker peak height.)

Thank you for your reply. Would you say these peaks corresponds to the upper marker? I was assuming they were larger un-tagmented regions, so I was planning to do a second size selection round with Ampure beads to remove these peaks. But if these are the markers then a second size selection might be unnecessary.

[kmcarr]

Originally Posted by ECorrales

Thank you for your reply. Would you say these peaks corresponds to the upper marker? I was assuming they were larger un-tagmented regions, so I was planning to do a second size selection round with Ampure beads to remove these peaks. But if these are the markers then a second size selection might be unnecessary.

What I am saying is that for those 5 lanes I mentioned there are 2 very closely spaced peaks, one labeled as a peak of ~1,260-1,270bp and the peak just after that labeled as the Upper marker. The software has incorrectly identified the later migrating peak as the Upper marker. In the TapeStation analysis software you should select the peak which is labeled ~1,270 and manually set that as the upper marker. If you do not correct the upper marker then the size calculation for these lanes will be incorrect.

[ECorrales]

Originally Posted by kmcarr

What I am saying is that for those 5 lanes I mentioned there are 2 very closely spaced peaks, one labeled as a peak of ~1,260-1,270bp and the peak just after that labeled as the Upper marker. The software has incorrectly identified the later migrating peak as the Upper marker. In the TapeStation analysis software you should select the peak which is labeled ~1,270 and manually set that as the upper marker. If you do not correct the upper marker then the size calculation for these lanes will be incorrect.

Oh, I see. Thank you so very much for clarifying it!