Chromosome conformation capture (Hi-C) analysis on lsr2, lsrL, and lsr2,lsrL deletion strains of S. venezuelae
Chromosome conformation capture (Hi-C) analysis on lsr2, lsrL, and lsr2,lsrL deletion strains of S. venezuelae
For preparation of S. venezuelae Hi-C contact maps, the standardized “small cultures” were setup. The cultures were incubated for 18 hours at 30˚C with shaking (180 rpm). After this time, the cultures were cross-linked with 1% formaldehyde for 7 minutes and 30 seconds, and blocked with 0.2 M glycine for 15 minutes. 1.2 ml of the cross-linked culture was centrifuged (1 minute, 5000 rpm at room temperature). The mycelium pellet was washed twice with 1.2 ml of TE buffer and resuspended in 0.6 ml of TE buffer.  The standardized “small cultures” were setup as follows: 5 ml of MYM medium was inoculated with 10^8 CFU, and the cultures were incubated for 18 hours at 30°C with shaking (180 rpm). Two vials, each containing 25 µl of the resuspended mycelium, were independently processed according to the modified protocol of Le*. Briefly, the cells were lysed using 0.5 µl of ReadyLyse solution (Invitrogen) at 37˚C for 30 minutes followed by the addition of 1.25 µl of 5% SDS solution and incubated at room temperature for 15 minutes. After cell lysis, the reaction was supplemented with 5 µl of 10% Triton X-100 (Sigma Aldrich), 5 µl of NEB3 buffer (New England Biolabs) and 10.75 µl of DNase-free water (Invitrogen), and subsequently incubated for 15 minutes at room temperature. The chromosomal DNA was digested with 2.25 µl of BglII restriction enzyme (New England Biolabs; 50000 U/mL) for 2 hours and 30 minutes at 37°C followed by the addition of 0.25 µl of BglII, and incubation for another 30 minutes at 37°C.  *  Chromosome Conformation Capture with Deep Sequencing to Study the Roles of the  Structural Maintenance of Chromosomes Complex In Vivo. Methods Mol. Biol. Clifton NJ 2004, 105–118  the Hi-C library preparation follow directly the protocol described by Le * The Hi-C libraries were PCR amplified according to the protocol64 with the number of amplification cycles increased up to 18. * Chromosome Conformation Capture with Deep Sequencing to Study the Roles of the  Structural Maintenance of Chromosomes Complex In Vivo. Methods Mol. Biol. Clifton NJ 2004, 105–118 