GSM1135051: ChIP-seq induced GFP-3Flag in neurospheres replicate 2; Mus musculus; ChIP-Seq
ChIP analysis was performed on 15x10^7 neural stem cells per condition (3XFlagFezf2IRESZsGFP, 3XFlagIRESZsGFP and untransduced MOCK) as described (Nelson et al., 2006) with modifications as detailed in the Supplemental Experimental Procedures. Briefly, cells were dissociated with 0.25% trypsin (Invitrogen), washed with PBS and chemically cross-linked with formaldehyde solution (1%). Cells were disrupted in lysis buffer (50 mM Tris, pH 8.0; 10 mM EDTA; 1% SDS;  and protease inhibitors) for 20 min on ice and sonicated with a Bioruptor to shear DNA into 200-700 bp fragments. Dynal magnetic beads (Sheep anti-mouse #M-28, Invitrogen, M2 FLAG) were preblocked with 5 μg of anti-FLAG M2 antibody per reaction (Sigma-Aldrich) overnight inverted at 4° C. Beads were then washed and resuspended in fresh 0.5% BSA diluted in PBS. Bead-antibody complexes were incubated at 4° C overnight with inversion. Beads were resuspended twice in low salt immune complex wash buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris-HCl, pH 8.1; and 150 mM NaCl), incubated for 5 min at 4° C, with rotation, and resuspended in LiCl immune complex wash buffer (0.25 M LiCl; 1% NP40; 1% deoxycholate; 1 mM EDTA; and 10 mM Tris-HCl, pH 8.1) twice and incubated for 5 min at 4° C, with rotation. Beads were then rinsed with ice-cold TE and DNA was eluted in 100 μl of elution buffer at 65º for 15 minutes. Reverse crosslinking was performed by incubating the ChIP DNA overnight at 65º. DNA was purified on QIAquick PCR purification columns (Qiagen) for use as template for Solexa library construction. ChIP-seq libraries prepared using standard Illumina protocols