title: GSM3711474: HeLa_CENP-C_IP; Homo sapiens; ChIP-Seq
library_construction_protocol: Libraries were constructed with the PacBio Standard Seq (v3) protocol. After cells were grown to ~80% confluency, they were harvested as described here (Bui et al 2012; Bui et al 2017), but with a few modifications. In short, cells were harvested, washed with PBS and PBS containing 0.1% Tween 20 (Sigma-Aldrich cat #P7949). Nuclei were released with TM2 (20 mM Tris-HCl, pH 8.0; 2 mM MgCl2; 0.5 mM PMSF) with 0.5% Nonidet P-40 (Sigma-Aldrich cat #74385). Afterwards, nuclei were washed with TM2 and dissolved in a total volume of 2 mL of 0.1 M TE (10 mM Tris-HCl, pH 8.0; 0.2 mM EDTA, 100 mM NaCl). Subsequently, chromatin was digested for 6 minutes with 0.25 U MNase (Sigma-Aldrich cat #N3755-500UN) and supplemented with 1.5 mM CaCl2. MNase reaction was quenched with 10 mM EGTA. All centrifugations were done at 1000 rpm at 4ºC. The cell or nuclei pellet was only tapped once to facilitate breaking them up. Supernatant was removed, and chromatin extracted overnight in low salt solution (0.5X PBS; 0.1 mM EGTA supplemented with a protease inhibitor cocktail (Roche cat #05056489001) N-ChIP chromatin bound to Protein G Sepharose beads (GE Healthcare cat #17-0618-02) were washed twice with ice cold 0.5X PBS and spun down for 1 minute at 4ºC at 800 rpm. The samples were used to prepare libraries for PacBio single-molecule sequencing as described in manufacturer's protocol (PacBio, Menlo Park, CA). Libraries were produced and loaded on ZMW chip either by diffusion or following size selection of the inserts (> 1000 bp) for all four samples.
attributes.tag: GEO Accession
attributes.value: GSM3711474