GSM6705500: MCF7 DpVp Mnase-seq; Homo sapiens; MNase-Seq
Cells were harvested at 80% confluency using a cell scraper, centrifuged at 125g for 5 min and washed once with cold phosphate-buffered saline. Cells were then resuspended in cold RSB buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 3 mM MgCl2) containing 0.5% Nonidet P-40, 1 mM PMSF and put on ice. The cell suspensions were homogenized by 20 strokes of pestle B in a Dounce homogenizer over 30 min on ice. Release nuclei were centrifuged in Sorvall X Pro centrifuge at 150g, 4ºC for 5 min, nuclei pellet was resuspended in cold RSB buffer supplemented with 1 mM PMSF. Before digestion, 1x10 6 nuclei in RSB buffer with 1 mM PMSF were supplemented with 2 mM CaCl2 and kept at 37ºC for 5 min. 5 units of Micrococcal nuclease (Nuclease S7 Micrococcal Nuclease, cat# 10107921001; Roche Applied Science) with 200 units ExoIII (cat# M0206S; New England Biolabs) were added to each nuclei aliquot and incubated for 10 min at 37ºC. The reaction was stopped by adding EDTA to a final concentration of 5 mM. Digested nuclei were centrifuged at 4,600g in a Sorvall Legend Micro tabletop centrifuge, supernatant S1 was removed and discarded. Pellet was resuspended in cold TE buffer, (10 mM Tris-HCl pH 8.0, 1 mM EDTA), gently pipetted and incubated on ice for 10 min. After centrifugation supernatant S2 was collected, pelleted material was resuspended in TE buffer, and after third centrifugation supernatant S3 was collected. Supernatants 2 and 3 were analyzed on 1% agarose gel in TAE buffer to confirm the release of DNA fragments of various length, then combined and treated with 1% SDS and 20 µg of proteinase K (New England Biolabs, #P8107S). The reaction was incubated for 1 h at 55ºC. DNA was extracted with phenol-chloroform-isoamyl alcohol, precipitated in 2.5 volumes of 100% alcohol supplemented with 50 mM sodium-acetate and left at -80ºC for overnight. Samples were centrifuged at 17,000g at a tabletop centrifuge, pellet was rinsed in 70% alcohol, air dried and dissolved in TE buffer (10 mM Tris-HCl, pH 8.0, 0.2 mM EDTA). Total DNA was sequenced on Illumina NextSeq2000 for a 100 bp pair-ended run to obtain 300 million reads per sample.