Instrument: Illumina HiSeq 2500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: DNA samples: Frozen cell pellets were resuspended in 1x IPOD lysis buffer (10 mM Tris HCl, pH 8.0; 50 mM NaCl) containing 1x protease inhibitors (Roche Complete Mini, EDTA free) and 52.5 kU/mL of ready-lyse (Epicentre); 600 uL per pellet (stationary phase cells were diluted 10x prior to lysis due to the much higher biomass of those pellets). We incubated the resuspended pellet for 15 minutes at 30 C, and then placed it on ice. We then sonicated the cells using a Branson digital sonicator at 25% power, using three 10 second bursts with 10 second pauses between bursts. The cells were maintained in a wet ice bath throughout sonication. We then performed a calibrated DNA digestion to sub-200 bp fragments, by adding to the sonicated lysates 60 ug RNase A (Thermo Fisher), 6 uL DNase I (Fisher product #89835), 5.4 uL 100 mM MnCl2, and 4.5 uL 100 mM CaCl2, and then incubating on ice. While the appropriate digestion time must be calibrated for each particular sample type and batch of DNase, 30 minutes of digestion proved appropriate for all samples here. Reactions were quenched after completion by the addition of 50 uL 500 mM EDTA (pH 8.0), typically yielding 50-200 bp fragments. Samples were clarified by centrifugation for 10 minutes at 16,9000xg at 4 C. After clarification, the lysate was mixed with 1 volume of 100 mM Tris base and 2 volumes of 25:24:1 phenol:chloroform:isoamyl alcohol, vortexed, and then incubated for 10 minutes at room temperature. After incubation, the sample was spun at 21,130xg for two minutes at room temperature, allowing formation of a white disc at the aqueous-organic interface enriched for protein-DNA complexes. The complete aqueous phases were removed and discarded, and the remaining disc washed again with 350 microliters TE (10 mM Tris, pH 8.0; 1 mM EDTA), 350 microliters 100 mM Tris base, and 700 microliters 24:1 chloroform:isoamyl alcohol. The resulting mixture was vortexed vigorously, and again centrifuged for 2 minutes at 21,130xg. All liquid was again removed, and the wash was repeated using 700 microliters TE and 700 microliters 24:1 chloroform:isoamyl alcohol. After vortexing, centrifugation, and removal of the final wash (exactly as above), any residual liquid was removed by wicking with a laboratory wipe (if any substantial pools of liquid were present). Finally, the interface was resuspended in 500 microliters of elution buffer (described above), vortexed vigorously, and reverse crosslinked overnight at 65 C. After allowing the samples to cool to room temperature, we then added 100ug of RNase A (Thermo-Fisher), incubated 2 hours at 37 C, then added 200ug of proteinase K (Fermentas) and incubated an additional 2 hours at 50 C. DNA was then recovered via standard phenol-chloroform extraction and ethanol precipitation, following protocols from (Ausubel, 1998). We used Glycoblue (Ambion) as a co-precipitant, NaCl as a precipitating salt (due to the presence of SDS in our solution), and washed with ice-cold 95% ethanol to avoid loss of low molecular weight DNA. RNA: RNA was harvested using Qiagen RNAProtect. RNA was isolated, and rRNA depletion was performed according to the Illumina Ribo-Zero rRNA Removal Kit for Bacteria. DNA samples: NEB Nebnext Ultra 2 DNA kit, but with a spin column replacing the first bead cleanup (see the accompanying paper for details).