title: yeast digital genomic footprinting
design: Digital DNase I libraries were constructed according to Illumina’s genomic prep kit protocol with minor modifications.  50 ng of purified DNase double-hit fragments (100-500 bp) were subjected to end repair by combining fractionated DNA, 1X T4 DNA ligase buffer (containing 0.1 mM ATP), 0.4 mM dNTP mix, 15 U T4 DNA polymerase, 5U DNA polymerase (Klenow, large fragment) and 50 U T4 polynucleotide kinase (all from New England Biolabs, Ipswich, MA).  The reaction mixture was incubated at 20 °C for 30 minutes and purified using a MinElute micro spin column (Qiagen).  Following end-repair, non-templated adenines were added to the 3’ ends of purified fragments with 1 mM dATP and 5 U Klenow fragment (3’- 5’ exo-) (NEB) and incubated at 37 °C for 30 minutes.   After column purification, Illumina adapters were ligated to the ends of DNA fragments in a 50uL reaction by combining 17.5 µL ‘A’-tailed fragments, 25 ul 2X Quick DNA ligase buffer (NEB), 2.5 µL adapter oligo mix (Illumina, Hayward, CA) and 5uL Quick DNA ligase (NEB) and incubating the mixture for 15 minutes at room temperature.  Ligations were purified with MinElute columns (Qiagen).    Adapter-ligated DNA fragments were enriched by amplification.  50 uL reactions consisting of 5 ul adapter-ligated DNA, 25 ul 2X Phusion High-Fidelity PCR Master Mix (NEB), 0.5 ul each of primer 1.1 and 2.1 (Illumina, Hayward, CA) and 19 uL nuclease-free water were assembled and cycled with the following thermal profile:  98 °C for 30 min., followed by 16 cycles of 98 °C for 10 min, 65 °C for 30 min. and 72 °C for 30 minutes with a final extension at 72 °C for 5 minutes.  Amplified libraries were purified with a MinElute micro spin column (Qiagen) and quantified using PicoGreen stain (Invitrogen) and a NanoDrop 3300 fluorospectrometer (Thermo Scientific, Waltham, MA).