GSM7286718: S2 2x (βA-globin full origin); Gallus gallus; OTHER
Exponentially growing cells were pulse-labeled with 5-Bromo-2'-deoxyuridine for 1h and four S-phase fractions (S1 to S4) were collected by flow cytometry or from an asynchronous cell population (AS). The cells were treated with lysis buffer (50 mM Tris pH 8.0; 10 mM EDTA pH 8.0; 300 mM NaCl; 0.5% SDS, 0.2 mg/ml of freshly added proteinase and 0.5 mg/ml of freshly added RNase A), incubated at 65°C for 2 h and stored at -20°C, in the dark. Genomic DNA was isolated from each sample by phenol-chloroform extraction and alcohol precipitation and sonicated four times for 30s each, at 30s intervals, in the high mode at 4°C in a Bioruptor water bath sonicator (Diagenode), to obtain fragments of 500 to 1000 bp in size. The sonicated DNA was denatured by incubation at 95°C for 5 minutes. We added monoclonal anti-BrdU antibody (BD Biosciences #347580) at a final concentration of 3.6 μg/ml in 1x IP buffer (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 150 mM NaCl, 0.5% Triton X-100, and 7 mM NaOH). We used 30 μl or 50 µl of protein-G-coated magnetic beads (from Ademtech #4342 or Thermo Fisher Scientific #10004D, respectively) per sample to pull down the anti-BrdU antibody. Beads and BrdU-labeled nascent DNA were incubated for 2-3 hours at 4°C, on a rotating wheel. The beads were then washed once with 1x IP buffer, twice with wash buffer (20 mM M Tris pH 8.0, 2 mM EDTA pH 8.0, 250 mM NaCl, 0.25% Triton X-100) and then twice with 1x TE buffer pH 8.0. The DNA was eluted by incubating the beads at 37°C for 2 h in 250 µl 1x TE buffer pH 8.0, to which we added 1% SDS and 0.5 mg/ml proteinase K. DNA was purified by phenol-chloroform extraction and alcohol precipitation and resuspended in 50 µl TE. Immunoprecipitated NS were amplified by whole-genome amplification (GenomePlex Complete Whole Genome Amplification kit #WGA2; Sigma) according to the manufacturer's recommendations to obtain sufficient DNA amount. Libraries were constructed with the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB #E7645S) following the manufacturer's instructions with minor modifications. For the adaptor ligation, undiluted adaptor and no size selection were used. The library amplification was performed using NEB-Next Multiplex Oligos for Illumina (NEB #E7710S) with nine different NEB-Next index primer and the NEB-Next Universal PCR Primer (NEB #E6861A) with three PCR cycles. Library purification was performed with the SPRI-select Reagent kit (Beckman coulter #B23317), and the final elution step was reduced to 33µl of 0.1×TE. The mean size of the library molecules determined on an Agilent Bio-analyser High Sensitivity DNA chip (Agilent technologies, #5067–4626) was 330-350 bp. Sequencing was performed on a NextSeq 500 Illumina sequencer with a High Output 150 cycles flow cell (paired-end reads of 75 bp) according to standard procedures. For each sample, 15-20 M of reads were generated.