GSM1847493: mESCs Nanog rep1; Mus musculus; ChIP-Seq
ChIPs were performed by using a modified published method (Ng et al., 2013). Briefly, Day (D)1 or D2 EpiLCs were cultured on a bacterial dish for 3h in the presence of 200(ng/ml) of Doxycycline. ESCs and induced EpiLCs were fixed with 1% formaldehyde (room temperature, 10 min), quenched with 1 vol. of 250 mM glycine (room temperature, 5 min), and rinsed with chilled TBSE buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA) twice before freezing in liquid nitrogen. After thawing on ice, 3e6 fixed cells were lysed with 1 mL 1% SDS Lysis Buffer (50mMTris-HCl pH8, 10mM EDTA, 1% SDS, Roche protease inhibitor cocktail on ice, 5 min) and then centrifuged (2,000 rpm, 15 min). Nuclear fraction was resuspended in 0.9 mL of dilution buffer (16.7mM Tris-HCl, pH8, 167mM NaCl, 1.2mM EDTA, 1.1% Triton X-100, 0.01% SDS, 0.2mM PMSF, 1X Protease Inhibitors). Samples were sonicated 10 times (30 s pulses with 30 s break interval) using a Bioruptor water bath sonicator (Diagenode). Chromatin extracts were then pre-cleared with Protein G Dynabeads and immunoprecipitated overnight with Protein G Dynabeads coupled with antibodies specific to Nanog (2μg). On the next day, beads were washed for 10 min at 4°C twice in each Low Salt (0.1% SDS, 1%, TritonX-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), High Salt (0.1% SDS, 1%, TritonX-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 300 mM NaCl), LiCl buffer (0.25 M LiCl, 1% NP40, 1% Na deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8), and TE buffer. After elution samples were digested with proteinase K and reverse crosslinked for 6 h at 68°C. 12ng of purified DNA from ChIPs was used for library preparation using Ovation Ultralow DR Multiplex System (0331, Nugen). Once prepared, library was size selected and sequenced using HiSeq2000 with single-end 50 nt read length.
GEO
GSM1847493