GSM2599092: MCF10CA1a Input; Homo sapiens; ChIP-Seq
DNA was extracted in cell lysis buffer (5mM PIPES pH8, 85mM KCL, 1% IGEPAL, protease inhbitors) on ice for 15 minutes, dounce homogenized 20x, and nuclear lysis buffer (50mM Tris-Cl pH8.1, 10mM EDTA, 1%SDS,  protease inhibitors) on ice for 30 minutes, and sonicated 12-14 minutes (140W, 10DF, 200c/b).  Samples were diluted  in RIPA and IP was carried out overnight at 4oC using the recommended antibody concentrations on 150ug of chromatin. Magnetic protein A/G beads were washed and added for an additional 4 hours. Beads were collected and washed 2x with RIPA minus protease inhibitors, and once with a second IP wash buffer (100mM Tris pH9, 500mM LiCl, 1% IGEPAL, 1% deoxycholic acid). Next the DNA was eluted using 50mM NaHCO3, 1%SDS 2 times 30 minutes on a shaker. Formaldehyde crosslinks were reversed using elution buffer plus .6M NaCl overnight at 67oC. ChIPseq libraries were prepared for sequencing using standard Illumina protocols using the TruSeq ChIP kit (Illumina #IP-202-1012) Barcoded libraries (Illumina TruSeq) were then loaded onto an Illumina HiSeq 1500 and single-end 100-base (SE100) sequencing was performed at the Advanced Genome Technologies Core Massively Parallel Sequencing Facility at UVM.
GEO Accession
GSM2599092