NS-seq Reads (MCF7)- Rep 1
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. For NS-seq, asynchronous cultures were grown to 70-80% confluency.         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 asynchronously         growing MCF7 cells (35-40% S-phase) with 15 ml of DNAzol (Invitrogen) following the         manufacturer’s directions and resuspended in DNA hydration buffer (Qiagen). Nascent strands         were prepared by adapting the protocol developed for replication initiation point mapping         (Gerbi and Bielinsky, 1997) for NS-seq. Replicative Intermediate (RI) DNA was enriched from         150 µg of genomic DNA by BND-cellulose chromatography (Sigma). Typically, 40 to 50 µg         (~25-30%) of starting material was recovered. The RI DNA was made single stranded by boiling         for 10 minutes and transferring to ice. The 5’ ends were phosphorylated with 50 units of T4         PNK for 1 hour at 37° C and the reaction was stopped by incubating 15 minutes at 75° C. The         fragments were digested with 100 units of Lambda-exonuclease (Fermentas) in glycine-KOH pH 8.8 buffer in a total         volume of 100 µl. The DNA:enzyme ratio was kept low to preserve the nascent strands because         Lambda-exo can lose specificity at high DNA:enzyme ratios and digest the RNA primer at the 5’ end         of DNA (Yang and Li 2013). Lambda-exo digested fragments were electrophoresed on a 1.5% UltraPure         LMP agarose gel and fragments in the range of 500-1500 nt were purified and resuspended in         Qiagen elution buffer. The concentration was determined by NanoDrop; 21-96 ng of DNA was         recovered for the replicates reported here, representing a ~500-2500 fold depletion of the         starting DNA. Nascent strand enrichment was determined by qPCR at the MYC locus using the         following primers: control locus: oMyc RT set 1-2 fwd, 5’-TTGCCAATTGCCTCTGGTTGAGAC-3’; oMyc         RT set 1-2 rev, 5’-GACTTTGCTGTTTGCTGTCAGGCT-3’; test primers: oMyc RT set 16-2 fwd, 5’-         TGAACCAGAGTTTCATCTGCGACC-3’; oMyc RT set 16-2 rev, 5’- AGAAGCCGCTCCACATACAGTCCT-3’. Single         stranded nascent strands were made double stranded with random hexamers and Klenow and         sonicated to a size of 100-600 bp. Illumina libraries were prepared using the NEBNext kit         following the manufacturer’s directions. 200-500 bp library fragments were size selected on         2% NuSieve agarose, gel purified and sequenced on the Illumina HiSeq         2000.