5 protocols
The Corona-Lite software (ABI) was used to align reads against the hg18 reference genome from UCSC (chr1-22,X,Y and mitochondrial genome). The first 32bp of each read was aligned allowing up to and including 3 mismatches.
SOLiD pipeline
DNA from individual immunoprecipiations (0.39 ng for ELK1, 0.96 ng for IgG, 6.68 ng for input - values for exp1; 3.4 ng for ELK1, 1.3 ng for IgG, 5 ng for input - values for exp2) was sent for sequencing according to the manufacturer's protocols (Life Technologies). Hardware: AB SOLiD System plus.
Nine million MCF10A cells (ATCC CRL-10317) were seeded into 150 mm dishes in media depleted of EGF.
After 48 hours cells were incubated for 10 minutes in 1% paraformaldehyde in 1xPBS; the crosslinking reaction was stopped by a 5 minute incubation in 0.125 M glycine in 1xPBS. Cells were washed with ice-cold 1xPBS and harvested into ice-cold 1xPBS containing the Complete protease inhibitor cocktail (Roche, 11836145001). Cells were washed with buffer I (10 mM HEPES pH 6.5, 0.5 mM EGTA, 10 mM EDTA, 0.25% TritonX-100), buffer II (10 mM HEPES pH 6.5, 0.5 mM EGTA, 1 mM EDTA, 200 mM NaCl) and lysed in 370 ul of lysis buffer (50 mM Tris pH 8.1, 10 mM EDTA, 1% SDS). Chromatin was sheared by eight 12 second rounds of sonication using Microson XL at power 9. At this stage, a 20 ul portion of the lysate was stored at -20 degrees Celsius until the proteinase K digestion step to be used as an input sample. Next, the total lysate from six plates was diluted 1:10 in IP buffer (16.7 mM TrisHCl pH 8.1, 0.01% SDS, 1.1% TritonX-100, 1.2 mM EDTA, 167 mM NaCl) and incubated overnight at 4 degrees Celsius with 8 ug of an appropriate antibody. For ELK1 (C-term RabMAb, Epitomics 1277-1). For IgG (Normal Rabbit IgG, Millipore, 12-370). Immunocomplexes were then incubated for one hour at 4 degrees Celsius with ProteinG-coated magnetic Dynal beads (Invitrogen, 100.04D) that were pre-blocked overnight at 4 degrees Celsius with sheared salmon sperm DNA (Sigma, D7656-1ML). Beads were washed twice with buffer TSEI (20 mM TrisHCl pH 8.1, 2 mM EDTA, 150 mM NaCl, 1% TritonX-100, 0.1% SDS), once with buffer III (10 mM TrisHCl pH 8.1, 250 mM LiCl, 1 mM EDTA, 1% IGEPAL, 1% DOC) and twice with buffer TE (10 mM TrisHCl pH 8.0, 1 mM EDTA); all washes were performed for 10 minutes at 4 degrees Celsius. Immunocomplexes were eluted into 150 ul of elution buffer (10 mM NaHCO3, 1% SDS) at room temperature. The input sample was diluted 1:5 with elution buffer. Crosslinks were reversed by addition of NaCl to the final concentration of 250 mM followed by an overnight incubation at 65 degrees Celsius. Protein in samples was digested by a 1 hour incubation with 1 ul of proteinase K (Roche, 031158887001) in 50 mM Tris-HCl pH 6.5 and 8.3 mM EDTA (final concentrations). DNA was purified using the QIAquick PCR purification kit (QIAgene, 28106) and used for sequencing.