Schematic comparison of standard single-stranded DNA library preparation and U selection. (1) Double-stranded ancient DNA molecules (gray lines) are dephosphorylated (not depicted) and heat-denatured. A deoxyuracil is denoted as a green circle. (2) Single-stranded adapter oligonucleotides carrying a biotinylated 3′ linker arm are ligated to the 3′ ends of the ancient molecules. The ligation products are then immobilized on streptavidin-coated magnetic beads (large circles). (3) An extension primer is hybridized to the adapter and Bst DNA polymerase is used to create a copy of the template strand. This reaction generates 3′ overhangs, which are subsequently removed in a blunt-end repair step using T4 DNA polymerase (data not shown). For U selection, T4 polynucleotide kinase is included in blunt-end repair to add a 5′ phosphate to the original template strand. (4a) In the standard protocol, a double-stranded adapter (blue) carrying a 5′ phosphate on one strand and a 3′ dideoxy block on the other strand is ligated to the newly synthesized strand using T4 DNA ligase. (5a) The library molecules are released from the beads by heat treatment. (4b) For U selection, a nonphosphorylated double-stranded adapter (blue) is ligated to the original template strand using T4 DNA ligase. (5b) The nick remaining in the opposite strand is removed in a fill-in reaction with the strand-displacing Bst polymerase. (6) If present, uracils are removed by uracil-DNA glycosylase, and the resulting abasic site is cut by endonuclease VIII, which leaves both 5′ and 3′ phosphates. The 3′ phosphates are removed with T4 polynucleotide kinase and the resulting 3′ hydroxyl groups are used to prime a strand-displacement polymerization reaction with Bst polymerase. (7) The supernatant now contains double-stranded library molecules originating from ancient DNA strands that carried a uracil, which (8a) are recovered in a separate tube, whereas (8b) the remaining library molecules are released by heat treatment.
