GSM8677820: output; Vibrio cholerae; ChIP-Seq
V. cholerae strains were initiated from freezer stocks and incubated at 30°C in LB broth rolling for ∼16 hours. The following day, cultures were subcultured into fresh LB to an OD600 of 0.08 and incubated rolling for 6 hours at 30°C. Cells were incubated with 1% paraformaldehyde at room temperature for 20 minutes shaking to crosslink DNA and proteins. A 1.2 molar excess of Tris (10 minutes, room temperature, shaking) was used to quench residual paraformaldehyde. Crosslinked cells were washed twice with TBS (25 mM Tris HCl, pH 7.5 and 125 mM NaCl) and then frozen at -80°C. Cells were then resuspended in lysis buffer (1x FastBreak cell lysis reagent (Promega), 50 μg/mL lysozyme, 1% Triton X-100, 1 mM PMSF, and 1x protease inhibitor cocktail; 100x inhibitor cocktail contained the following: 0.07 mg/mL phosphoramidon (Santa Cruz), 0.006 mg/mL bestatin (MPbiomedicals/Fisher Scientific), 1.67 mg/mL AEBSF (Gold Bio), 0.07 mg/mL pepstatin A (DOT Scientific), 0.07 mg/mL E64 (Gold Bio) suspended in DMSO) to an OD600 of 50.0. These resuspensions were incubated rocking at room temperature for 20 minutes to permit cell lysis. Lysates were then sonicated to shear DNA into 200-300bp fragments (sonication was performed with a Fisherbrand Model 705 Sonic Dismembrator 6 times for 10 seconds at 1% amplitude, resting on ice for at least 30 seconds between each sonication). The sonicated lysates were then clarified by centrifugation and diluted 1:5 in Immunoprecipitation (IP) Buffer (50 mM HEPES NaOH pH 7.5, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100). An “input DNA” sample was collected from this diluted lysate. 1 mL of this diluted lysate was then added to Pierce anti-DYKDDDDK magnetic agarose equilibrated in IP buffer and then incubated at room temperature with end-over-end mixing. After two hours of incubation, a magnetic separation rack was used to facilitate removal of the supernatant. The agarose was washed twice with IP Buffer for 1 minute, once with Wash Buffer 1 (50 mM HEPES NaOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 500 mM NaCl, and 0.1% SDS) for 5 minutes, once with Wash Buffer 2 (10 mM Tris HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, and 1% Triton X-100) for 5 minutes, and once with Buffer TE (10 mM tris pH 8.0 and 1 mM EDTA) for 1 minute. SDS elution buffer (50 mM Tris HCl pH 7.5, 10 mM EDTA, and 1% SDS) was used to elute protein and DNA off the magnetic agarose (30 minutes at 65°C). Supernatant was collected and digested for 2 hours with 20 μg Proteinase K in SDS Elution Buffer at 42°C. 6 hours of incubation at 65°C was then used to reverse crosslinking. Both the input and the output samples were then purified via silica spin columns to remove non-DNA components. Libraries were prepared using the New England Biolabs NEBNext Ultra II DNA Library Prep Kit for Illumina protocol (catalog no. E7645L) and analyzed by Agilent 4200 TapeStation. Libraries were pooled and loaded on a NextSeq 1000/2000 P2 Reagents (100 Cycles) v3 flow cell (catalog no. 20046811) configured to generate 2x61 nt paired end reads. Demultiplexing was performed with bcl2fastq, version 2.20.0.