title: GSM3676128: dKO+BraGFP_cells_d5_nodox (ChIP-seq); Mus musculus; ChIP-Seq
library_construction_protocol: The protocol was modified from Schmidt et al., according to Singh et al. Briefly, ProteinG beads (50 μl per sample) were incubated with 5 μg of Anti-GFP Antibody (Abcam, ab290) overnight at 4°C. Cross-linking was performed with 2 mM disuccinimidyl glutarate (DSG, Thermo Fischer, 20593) in the SolutionA (50 mM Hepes, 100 mM NaCl, 1 mM EDTA and 0.5 mM EGTA) for 15 min followed by 1% formaldehyde (Sigma Aldrich, F8775) for additional 10 min. The reaction was stopped by adding 2.5 M Glycine for 5 min. Cells were lysed in Lysis buffer 1 (50 mM Hepes, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40 and 0.25% TritonX in H2O, pH7.5) with protease inhibitors (PI, Complete™ Protease Inhibitor Cocktail, Roche) for 10 min on a rotating wheel at 4°C. Next, pellets were incubated for 5 min at 4°C with Lysis buffer 2 with PI (10 mM Tris, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA in H2O, pH8), followed by resuspension in Lysis buffer 3 with PI (10 mM Tris, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% Lauroylsarcosine in H2O, pH8). The DNA was sonicated with the Bioruptor (Diagenode), at high amplitude for 10-12 cycles of 30 s ON/30 s OFF. TritonX was added to final concentration of 1%. 10% of the resulting lysate was kept as input control. Beads coupled to antibodies incubated with sonicated chromatin at 4°C overnight. Next, beads were washed on ice ten times with RIPA buffer (50 mM HEPES, 500 mM LiCl, 1 mM EDTA, 1% NP40 substitute, 0.7% Na-Deoxycholate in H2O, pH7.6) and once with TBS. DNA was removed from the beads with Elution buffer (50 mM Tris, 10mM EDTA, 1% SDS in H2O, pH8) for 6-18 h at 65°C. The supernatant was mixed with one volume of TE buffer (10 mM Tris, 1 mM EDTA in H2O, pH7.4), incubated with RNAse A at 37°C for 1 h, and ProteinaseK at 55°C for 2 h. Phenol-chloroform extraction of the DNA was performed and pellet  was resuspended in 50 μl of 10 mM Tris-HCl pH8. Libraries were prepared with NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs), size selected on a 1.3% agarose gel by cutting out fragments between 200-650 bp of size and extracted from the gel using QIAquick gel extraction kit (Qiagen).
attributes.tag: GEO Accession
attributes.value: GSM3676128