Klenow 3 to 5 exo minus

Genomic DNA was sheared to 300–600 bp size using a focused ultra-sonicator from Covaris (Woburn, MA), size purified by agarose gel electrophoresis, and analyzed on a DNA Chip using a 2100 bio-analyzer (Agilent Technologies, Inc.) to verify size distribution. DNA was end-repaired (End-It kit, Epicenter Biotechnologies), an A-overhang was added (Klenow 3′ to 5′ exo minus, NEB), and ligated to Illumina pair-end adapter sequences (Illumina). The ligated libraries were size selected (600±50 bp), PCR amplified for 10 cycles, and purified with SPRI beads (Agencourt AMPure XP; Fisher Scientific). Pair-end library quality was verified on a bio-analyzer as described above. Each library was sequenced on three lanes on the Illumina HiSeq 2000 yielding about 10⁹, one hundred bp pair-end reads corresponding to about 30× coverage for each S phase library.

Sequencing libraries were prepared using the following procedure, as previously described³⁵ . The concentration of immunoprecipitated DNA was measured using Qubit dsDNA HS Assay kit (Thermo Fisher Scientific). Barcoded sequencing libraries were generated from 1 ng of immunoprecipitated DNA⁵² . Briefly, immunoprecipitated DNA was end repaired using T4 DNA polymerase, T4 PNK, and DNA polymerase I Large (Klenow) fragment (New England Biolabs, Ipswich, MA). A single adenosine was added to the 3′ end of fragments using Klenow (3′ to 5′ exo minus, New England Biolabs) and then adapters containing multiplexing barcodes were ligated using T4 Quick DNA ligase (New England Biolabs). Adapter ligated DNA fragments between 200 and 500 bp were gel purified and PCR amplified with 16 cycles using Phusion DNA polymerase (ThermoFisher Scientific). Quality of libraries was examined using Agilent 2100 Bioanalyzer (Agilent Santa Clara, CA). Equimolar amount of each library was mixed and 50 bp single-end sequenced in High Output (Standard) v3 Illumina HiSeq 2000 (Harvard Bauer Center Core Facility, Cambridge, MA).