title: 3C-seq analysis of Acinetobacter baumannii AB5075 in the presence and absence of H-NS-39
design: 3C-seq analysis of Acinetobacter baumannii AB5075 in the presence and absence of H-NS-39
library_construction_protocol: A. baumannii AB5075 is a clinical strain isolated from an infected tibia.Jacobs, A. C. et al. AB5075, a Highly Virulent Isolate of Acinetobacter baumannii, as a Model Strain for the Evaluation of Pathogenesis and Antimicrobial Treatments. (2014) doi:10.1128/mBio.01076-14. Cells were grown in liquid LB medium at 37 degrees with shaking. Our procedure was based on the protocol of Rashid et al. Nuclease free water was used throughout. Two biological replicates were done for each strain background. We began with a 10 ml culture of A. baumannii grown to an OD600 of 0.5. Cells were recovered by centrifugation and resuspended in 4 ml of PBS before addition of 16 ml ethanol. The samples were incubated on a roller bench at 30 rpm for 10 minutes at 4 ºC. After recovery by centrifugations, cells were washed with 20 ml of ice-cold PBS and resuspended in 40 ml of 3 % (v/v) formaldehyde diluted in PBS. The mixtures were again placed on the roller bench at 4 ºC and left for 1 hour. Glycine (400 mM) was used to quench the reaction during a further 15 minute roller bench incubation. Cells were then recovered, resuspended in 5 ml of TE, and divided into 1 ml aliquots, pelleted, and flash frozen using liquid N2. For cell lysis, we resuspended a cell aliquot in 50 μl of TE containing 0.5 μl of diluted lysozyme (made of up 0.5 μl Ready-Lyse lysozyme, 4 μl TE and 0.5 μl 1M NaCl). The resuspension was then incubated at 37 ºC for 15 minutes. The lysed cells were diluted to a final volume of 54 μl with TE before being mixed with 136 μl of water, 25 μl 10 % (w/v) Triton X-100 and 25 μl 10x rCutsmart restriction digest buffer.   Rashid, F. Z. M., Detmar, L. & Dame, R. T. Chromosome Conformation Capture in Bacteria and Archaea. Methods in Molecular Biology vol. 2516 (Methods Mol Biol, 2022).  We then added 10 μl of NlaIII (10 U/μl) for 3 hours at 37 ºC. Reactions were stopped by adding 12.5 μl of 10 % (w/v) SDS and incubating for a further 20 minutes. Digested nucleoprotein was centrifuged for 1 hour, at 4 ºC and 25,000 x g. The DNA was then pelleted, resuspended in 200 μl water, and its concentration determined. Between 1-3 μg of digested nucleoprotein was diluted to 895 μl using water and 100 μl of 10x T4 DNA ligase buffer was added alongside 5 μl of 20 mg/ml BSA and 2 μl T4 ligase (2000U/μl). Multiple reactions were set up to use all of the chromatin and incubated at 16 ºC for 16 hours. The incubation was then transferred to a Thermoixer for 1 hour at 25 ºC with shaking at 450 rpm. The reaction was stopped with 20.5 μl of 0.5 M EDTA. After ligation, RNA was removed with 16.6 μl of 10 mg/ml RNAse A for 30 minutes at 37 ºC with shaking at 450 rpm.  We then added 12.5 μl of 20/mg/ml proteinase K, and 120.8 μl of 5 M NaCl, for 16 hours at 65 ºC with shaking at 450 rpm. Samples were then pooled in a 15 ml Falcon tube before addition of 1 volume of 25:24:1 phenol:chloroform:isoamyl alcohol followed by vigorous mixing. The aqueous and organic layers were separated by centrifugation at 13,000 x g for 10 minutes at 4 ºC. The upper aqueous layer was transferred to a new Falcon tube and the process was repeated. Next, 0.1 volume of 1 M sodium acetate pH 8.0, 0.025 volumes and 5 mg/ml glycogen, and 2.5 volumes of ice-cold ethanol were added before incubation at -20 ºC overnight. The DNA was recovered by centrifugation at 25,000 x g and 4 ºC for 20 minutes and the pellet washed with 0.5 ml ice cold 70 % (v/v) ice cold ethanol. The DNA was again pelleted and then dried in a 50 ºC incubator. Each dried pellet was dissolved in 31 μl of water in a Thermomixer at 60 ºC at 1000 rpm. After checking ~6 μl of the library using agarose gel electrophoresis, the remaining sample (which should contain least 1 μg of DNA) was mixed with 60 μl of NEBNext sample purification beads in a low retention 1.5 ml tube. After mixing and incubation for 5 minutes at 25 ºC beads were washed twice with 200 μl of 70 % (v/v) ethanol. The DNA was then eluted with 25 μl of TE and the concentration determined. A minimum of 100 ng was then used to generate the final library using the NEBNext Ultra II FS DNA PCR-free library prep kit (high-input protocol).