Hi-C of Klebsiella Pneumoniae
The protocol for the Hi-C assay was performed as previously described. Bacteria cultured to the early logarithmic phase were collected. A final concentration of 3% fresh formaldehyde was used to crosslink the bacteria at room temperature (RT) for 10 min. The formaldehyde was quenched by incubation with a final concentration of 0.375 M glycine at 4 C for 20 min. The cross-linked bacterial samples were resuspended in Tris-EDTA (TE), followed by addition of lysis buffer (50 U/L). Lysis was performed at 37 C in an incubator for 10 min and followed by addition of SDS to a final concentration of 0.5% to accelerate lysis and the samples were left for a further 10 min at RT. 10% Triton X-100 was used to terminate the lysis reaction and the samples were incubated at RT for 10 min. After centrifugation, the supernatant was discarded and the DNA that collected in the pellet was digested by adding the digestion system (10  NEB buffer 2.1 and 100 U of Sau3AI) and placing in an incubator at 37 C. The end-complementation system (10  2.0 NEB buffer, Klenow, 10 mM dATP/dTTP/dGTP, 5 mM biotin-16-AA-2'dCTP) was added and the reaction was left at RT for 4 h. Flat-end ligation was performed using T4 DNA ligase at 16 C overnight. After ligation, RNase was added to remove residual RNA and 200 mg/mL proteinase K (Thermo) was added before incubation at 65 C for 5 h. DNA was purified and recovered using AMpure beads according to the manufacturers protocol. The samples were sonicated using Covaris Snol Lab7 and the DNA was interrupted to ~400 bp length before being purified and recovered using AMpure beads. The target biotinylated fragments were captured using DynabeadsR MyOneTM Streptavidin C1 (Thermo Fisher Scientific). Hi-C libraries were constructed using the NEBNextR UltraTM II DNA Library Prep Kit for Illumina (NEB) according to the manufacturers protocol. Fragment lengths from 400 to 600 bp were analyzed on an Illumina HiSeq X Ten platform (San Diego, CA, USA) with double-end sequencing and two replicates per set of samples.