title: Sequencing of GM06990 S1 phase
design: Illumina sequencing libraries were constructed from the random-primed BrdU-DNA material according to standard procedures. Illumina’s genomic prep kit protocol was used with minor modifications (Illumina, Hayward, CA): 10–50 ng of purified random-primed products 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 units T4 DNA polymerase, 5 units DNA polymerase (Klenow, large fragment), and 50 units T4 polynucleotide kinase (NEB, Ipswich, MA). The reaction mixture was incubated at 20°C for 30 minutes and purified using a MinElute micro spin column (Qiagen, Valencia, CA). Following repair, non-templated adenines were added to the 3' ends of purified fragments with 1 mM dATP and 5 units Klenow fragment (3'-5' exo-minus) (NEB) and incubation at 37°C for 30 minutes.    After column purification, adapters (Illumina) were ligated to the ends of DNA fragments in a 50 µl reaction by combining 17.5 µl ‘A-tailed’ fragments, 25 µl 2X Quick DNA ligase buffer (NEB), 2.5 µl adapter oligo mix (Illumina), and 5 µl Quick DNA ligase (NEB) and incubation for 15 minutes at room temperature. The adapter-ligated material was then purified with MinElute columns (Qiagen) and enriched by a limited amplification: 50 µl reactions consisting of 5 µl adapter-ligated DNA (equivalent to about one tenth of the ligated material), 25 µl 2X Phusion High-Fidelity PCR Master Mix (NEB), 0.5 µl each of primer 1.1 and 2.1 (Illumina), and 19 µl 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 min with a final extension at 72°C for 5 minutes.    Amplified libraries were purified with a MinElute micro spin column (Qiagen) and size-fractionated using agarose electrophoresis. Fragments in the 250–500 bp range were purified from the gel using the Qiagen gel extraction kit according to the manufacturer’s recommendations and quantified with PicoGreen staining. One sixth of each enriched library was sequenced in a single lane on a Genome Analyzer II (Illumina) using standard sequencing-by-synthesis technology.