GSM8646393: MN-ORC5 cloneA13 10min Rep3 [ORC5 ChECseq]; Homo sapiens; OTHER
For each ChEC experiment, cells were grown in five 10-cm plates. Cells were pelleted at 600 × g at 4 °C for 3 min and washed three times with Washing Buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.1 mM EGTA, 0.5 mM spermidine, 0.1% BSA, 1 × Roche cOmplete EDTA-free mini protease inhibitors, 1 mM PMSF), centrifuging as above between washes. Cells were resuspended in NE Buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 0.1 mM EGTA, 0.5 mM Spermidine, 0.1% Triton X-100, 20% Glycerol, 1 × Roche cOmplete EDTA-free mini protease inhibitors) and rotated at 4 °C for 10 min. After centrifugation, cells were washed once each with Washing Buffer and Wash Buffer without protease inhibitor and resuspended in Wash Buffer without protease inhibitor. CaCl2 was added to 2 mM and ChEC digests were performed at 25 °C. At each time point, a 150-μl aliquot of the digest was transferred to a tube containing 150 μl 2 × stop buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 µg/mL RNase A, 20 µg /mL Glycogen) and incubated at 37 °C for 30 min. After adding 0.1% SDS, protein was digested with 100 μg proteinase K at 55 °C for 30 min. DNA was extracted one each with an equal volume of phenol and phenol/chloroform/isoamyl alcohol. DNA in aqueous phase was size-selected with 0.45x vol SPRIselect beads and then precipitated with 200 mM NaCl and 2.5 volumes 100% ethanol. Pellets were washed once with 1 ml 70% ethanol, dried, and resuspended in 30 μl 0.1 × TE buffer, pH 8.0. Sequencing libraries was prepared by NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645S).