DNA repair and end-prep

In the first step, DNA repair was achieved through the utilization of a cocktail of enzymes (NEBNext FFPE DNA repair Mix) designed specifically to repair breaks in the DNA. This cocktail is able to repair nicks, gaps (up to ten nucleotides), blocked 3′-ends, oxidized bases, and deamination of cytosine to uracil. Afterwards, the DNA ends are repaired through the Ultra II End-prep enzyme mix, which enhances the attachment of the DNA barcodes. It is important to note that BACs are extremely sensible to physical sharing. For this reason, throughout this protocol, we mixed the samples always by manual flicking to avoid to pipette and vortex. DNA (1.2–1.5 µg) were transferred into a 1.5 ml DNA LoBind tube and the volume adjusted to 48 µl with Nuclease-free water and mixed thoroughly by inversion or flicking. We added to the DNA following exactly this order directly in the LoBind tube, 3.5 µl NEBNext FFPE DNA Repair Buffer, 2 µl NEBNext FFPE DNA Repair Mix, 3.5 µl Ultra II End-prep reaction buffer, and 3 µl Ultra II End-prep enzyme mix. The samples were incubated at 20 °C for 5 min and 65 °C for 5 min. During the incubation we prepared 500 µl of fresh 80 % ethanol in Nuclease-free water. We added 60 µl of resuspended AMPureXP beads to the end-prep reaction and mixed by flicking the tube. The samples were incubated on a Hula mixer (rotator mix) for 15 min at room temperature, then spun down briefly and placed on a magnetic rack. We kept the LoBind tube on the rack and pipetted off the supernatant, then the beads were washed twice with 200 µl of 80% ethanol. The samples were spanned down and placed back on the magnet. The residual ethanol was removed and the samples left to air dry for 60 s. The samples were resuspend by flicking in 25 µl Nuclease-free water. To enhance elution, we incubated the samples for 20 min at 37 °C by using a thermocycler.

The pellet was spun down and the sample placed on a magnetic rack until the eluate is clear and colorless, we removed and retained 25 µl of eluate into a clean 1.5 Eppendor DNA LoBind tube.

One microliter of end-prepped DNA was measured using a Qubit fluorometer (recovery aim > 200 ng).

We then proceeded with the naive barcoding step.