LexoG0 Reads (MCF7)- Rep1
MCF7 breast cancer cells were obtained from ATCC and grown in Dulbecco’s         Modified Eagle medium with 10% fetal calf serum supplemented with 100 U/mL penicillin and         100 µg/mL streptomycin. Cells were synchronized in G0 by plating at 50% confluency and serum         starving for 24 hours. The proportion of cells in S-phase was determined by FACS analysis         (BD FACSCalibur). Three biological replicates were performed. For each, genomic DNA was         purified from serum starved cells (<= 9.6% S-phase) with 15 ml of DNAzol (Invitrogen)         following the manufacturer’s directions and resuspended in DNA hydration buffer (Qiagen).         150 µg of DNA was sonicated to a size range of 200 bp to 10 kb in a Biorupter Standard         (Diagenode) and purified with Agencourt Ampure XP beads (Beckman Coulter). In order to         investigate the genome-wide, nascent strand independent Lambda-exo biases in the genomic DNA it         is important to avoid enriching the small amount of contaminating S-phase DNA that may be         present. Fragmentation of the DNA breaks any long, RNA-primed nascent strands associated         with replication forks into smaller fragments, ensuring that short RNA protected fragments         (if present) are distributed throughout the genome rather than only near origins, which         prevents origin sequences from being accidentally enriched. The fragmented DNA was made         single stranded by boiling for 10 minutes, and transferring to ice. The 5’ ends were         phosphorylated with 50 units of T4 Polynucleotide Kinase (T4 PNK; New England Biolabs) for 1         hour at 37° C. The reaction was stopped by incubating 15 minutes at 75° C. Following         phenol:chloroform extraction and ethanol precipitation, the phosphorylated fragments were         digested with 100 units of Lambda-exonuclease (Fermentas) in glycine-KOH pH 9.4 buffer in a         total volume of 100 µl. The reaction was stopped by incubating 15 minutes at 75° C before         the samples were phenol:chloroform extracted and ethanol precipitated. Lambda-exonuclease         digested fragments were electrophoresed on a 1.5% UltraPure LMP agarose (Invitrogen) gel.         Fragments in the range of 500-1500 nt were then purified by melting at 65° C for 10 min         before sequential extraction with phenol, phenol chloroform, and chloroform, followed by         resuspension in 10 µl elution buffer (Qiagen). The concentration was determined by NanoDrop         (Thermo Scientific). Lambda-exonuclease typically depletes the input DNA 1000-fold at this         step. The purified single stranded fragments were made double stranded with random hexamers         and Klenow (New England Biolabs), then sonicated to a size of 100-600 bp. Illumina libraries         were prepared using the NEBNext kit (New England Biolabs) following the manufacturer’s         directions. 200-500 bp library fragments were size selected on 2% NuSieve agarose (Lonza)         and were gel purified (Qiagen). Libraries were sequenced on the Illumina HiSeq 2000         platform.