GSM1567502: In_Vitro_occupancy; Mus musculus; OTHER
BAC clones of Oct4, Sox2, Nes, Pax6, Olig2 and Sox1 and surrounding regions were purified from the RPCI-23/24 library (Children’s Hospital Oakland Research Institute, Oakland, CA, USA). BACs were evenly combined into a total pool of 200ng and nicked to add biotin tags, using Roche’s nick translation kit (Roche 10976776001) and 40uM biotin-dUTP (Axxora ENZ-42811).  100ng of biotin-nicked BAC DNA was concentrated and resuspended with 10μg of Cot-1 DNA (Invitrogen 18440-016).  This mixture was denatured, suspended in 2x hybridization buffer (1.5 M NaCl, 40 mM sodium phosphate buffer (pH 7.2), 10 mM EDTA (pH 8), 10x Denhardt’s, 0.2% SDS), then incubated at 65°C for 6 hours.  1μg of  adaptor-ligated mononucleosome DNA was denatured, also suspended in 2x hybridization buffer, then combined with the BAC DNA mixture and incubated at 65°C for a further 70-80 hours.  The hybridization solution was bound to 100μL of streptavadin-coated beads (Invitrogen 11205D), suspended in 150μL of washing/binding buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 8) and 1 M NaCl) incubated for 30 minutes, then stringently washed with one wash of 1x SSC, 0.1% SDS buffer at room temperature, and three washes of 0.1x SSC, 0.1% SDS buffer at 65°C.  The DNA was eluted with 100μL of 0.1 M NaOH, neutralized with 100μL of 1M Tris-HCl (pH 7.5), then desalted and cleaned using two spin chromatography columns (Biorad 732-6223). Single-stranded enriched DNA was then amplified and isolated using a high-fidelity polymerase kit (NEB E0553S) according to recommended reaction conditions, and using ABI SOLiD library primers (Invitrogen 4464411). Test reactions of 25μL were cycle titrated to determine the appropriate number of rounds of amplification, then bulk reactions of 2x 50μL were amplified.  Theses products were loaded onto a 3.3% NuSieve agarose gel in TAE and excised and purified. DNA libraries were prepared for sequencing using SOLiD 4 standard library preparation protocols