title: Illumina HiSeq 2000 sequencing; ETEC H10407 ChIP-Seq
design: ETEC H10407 ChIP-Seq
library_construction_protocol: 5mL cultures were set up in M9 minimal media, supplemented with 0.4% fructose and 0.2% casamino acids and grown for 48 hours at 37 degrees C/250rpm.  Overnight cultures were subcultured 1:100 and grown for 7 hours at 37 degrees.   Cultures were crosslinked by adding formaldehyde to a final concentration of 1%, incubated for 20 minutes at room temperature, and quenched by adding glycine to a final concentration of 0.5 M.  Cells were washed once in original culture volume of 1X TBS then washed again in 1 mL of 1X TBS.  The supernatant was removed and the pellet was stored at -20degrees C. Extraction and Library construction occur in the same protocol: Pelleted cells were resuspended in FA lysis buffer containing 2 mg/mL of lysozyme.  Samples were incubated for 30 minutes at 37degrees C, chilled on ice for at least 5 minutes, then sonicated for 30 minutes, with 30 s pulses followed by 30 s off.  Samples were spun at maximum speed in a microcentrifuge, the supernatants were removed and diluted to 1 mL lysate per 20 mL original culture volume.  Samples were immunoprecipitated according to the following procedure:  for every 800 uL of chromatin, 25 uL of protein A beads (50% TBS slurry), and 2 uL anti-FLAG antibody (M2) were added.  Samples were incubated on rotisserie rotater for 90 minutes at room temperature.  Samples were then transferred to Spin-X columns (Corning) and washed twice with FA lysis buffer (150mM NaCl), 3 minutes each wash, rotating at room temperature.  The samples were spun at 4000 rpm after each wash.  The samples were then washed twice in the same way with 10 mM Tris-HCl, pH 7.5.   The immunoprecipitated DNA, still attached to protein A beads, were then treated with Quick Blunting Kit (NEB):  The samples were resuspended in the following mixture: 10ul 10x quick blunting buffer, 10ul dNTP mix (kit supplied), 80ul dH2O, 2ul blunt enzyme mix.  The samples were incubated at room temperature, while rotating, for 30 minutes.  The samples were then washed twice with FA lysis buffer (150mM NaCl), as described above, and then washed twice with 10 mM Tris-HCl, pH 8.0.  The samples were then A tailed, using Klenow (3-5exo), from NEB:  The samples were resuspended in the following mixture: 10ul 10X NEBuffer 2, 2ul 100mM dATP (NEB), 88ul dH2O, 2ul Klenow.  The samples were incubated at 37 degrees, while rotating, for 30 minutes. The samples were then washed twice with FA lysis buffer (150mM NaCl), as described above, and then washed twice with 10 mM Tris-HCl, pH 7.5.  Illumina adapters were then ligated to the samples using the NEB Quick Ligation kit.  The samples were resuspended in the following mixture:  100ul 1x ligase buffer (50ul 2x buffer + 50ul dH2O), 1 ul bracoded adapter, 4ul quick ligase.  The samples were then incubated at room temperature, while rotating for 15 minutes.  After incubation, the samples were washed, as decribed above, in the following buffers: FA lysis buffer; FA lysis buffer+500 mM NaCl; ChIP wash buffer; TE (10 mM Tris pH 8.0, 1 mM EDTA). Spin-X columns were transferred to a new microcentrifuge tube and 100 uL of ChIP elution buffer was added.  Samples were incubated for 10 minutes at 65 degrees C then spun for 1 min at 1500 x g.  Eluted DNA was then transferred to a new microcentrifuge tube and incubated for 10 minutes in a boiling water bath to de-crosslink samples.  The samples were phenol extracted and ethanol precipitated.  The samples were resuspended in 11ul.  To determine cycle numbers for library amplification of each sample, 1ul ChIP DNA was used as a template (diluted 1/8 to fill 3 wells) for Real Time PCR (7500 fast, ABI) with Illumina primers 1.1 and 2.1 (1uM ea).  The manual ct was set to 0.1 and '3' was added to the cycle number to determine the amount of cycles for library amplification.   The ChIP DNA was used as a template, using the determined cycle number of : 95 degrees for 30 seconds, 60 degrees for 30 seconds, 72 degrees for 30 seconds. The following 50ul PCR reaction was used:  5ul 10x taq buffer,  8ul ChIP DNA with ligated adapters, 1.0 ul Illumina primer 1.1 (100uM), 1.0 ul Illumina primer 2.1 (100uM), 0.4 ul dNTP mix (25mM each), 1.5 ul taq, 33.6 ul dH2O.  The libraries were checked on an 8% non-denaturing acrylamide gel.  The smear from 200bp  to 500bp was cut out and gel purified.  The bar-coded libraries were quantified using Qubit and pooled at equimolar concentrations.
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: untagged