title: GSM8771725: ES114, WT, rep1, input; Aliivibrio fischeri; ChIP-Seq
library_construction_protocol: For the whole genome sequencing (WGS; input) samples, a 3 ml aliquot was pelleted before crosslinking and gDNA was extracted using the ThermoFisher GeneJet DNA Purification Kit. The DNA was diluted to 10 ng/µl in 100 µl of elution buffer and sonicated at 4°C twice with a Qsonica Q800R2 Water Bath Sonicator at 20% amplitude with continuous pulses. WGS samples were stored at -20°C until library preparation. The remaining cultures (47 ml) (immunoprecipitation (IP) samples) were crosslinked by adding 1% formaldehyde (Fisher Scientific) and rocked for 30 min at room temperature. The reactions were quenched with 0.5 M glycine and rocked for 5 min at room temperature. Cultures were pelleted at 4°C, washed 2 times with 1 ml cold 1x TBS, frozen with liquid nitrogen, and stored at -80°C until needed. During lysis, pellets were resuspended in ChIP Solution A (12.5 mM Tris pH 8, 12.5 mM EDTA, 62.5 mM NaCl, 25% sucrose, 1 mM PMSF) with 1 mg/ml lysozyme for 45 min at 37°C. An equal volume of 2x IP Buffer was added to the solution followed by 1 mM PMSF and 1x Protease Inhibitor Cocktail. Samples were sonicated twice for 20 min at 70% amplitude with 10 sec on/off pulses and then centrifuged to clear cell debris. Sonicated lysates were pre-cleared by rotating for 1 h at 4°C using 50 µl washed Protein A Magnetic Sepharose beads (Cytiva #28967062). Pre-cleared lysates were incubated with 4 µl anti-LuxR (V. campbellii) and rotated overnight at 4°C. The next day, 50 µl Protein A magnetic beads were added to the lysate-antibody mixtures and rotated for 1 h at 4°C. The resin was washed 3 times with 1x IP Buffer, 3 times with Wash Buffer, once with 1x TE, and eluted with TES at 65°C for 20 min. Crosslinks were reversed at 65°C overnight and then treated with 0.2 mg/ml RNaseA (NEB #T3018L) and 0.2 mg/ml Proteinase K (NEB #P8107S). DNA was extracted using a phenol:chloroform:isoamyl alcohol (25:24:1) mixture with ethanol precipitation, then purified using AmpureXP beads (Beckman Coulter, Inc.). WGS and IP libraries were prepared for Illumina NextSeq550 high-throughput paired-end sequencing using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB #E7645)
identifiers.namespace: GEO
identifiers.id: GSM8771725_r1