GSM6876594: ZM4, RM, rep2; Zymomonas mobilis; Hi-C
Cells were then crosslinked with 3% fresh formaldehyde for 30 min at room temperature, followed by 30 min at 4 °C. Formaldehyde was finally quenched with 0.375 M glycine for 20 minutes at 4 °C. Fixed cells were collected, frozen in liquid nitrogen, and stored at -80 °C until use. 1 × 10^9 cells were suspended in 100 μl TE with 2 μl of Ready-Lyse lysozyme (Epicentre), and incubated at room temperature for 20 min before being treated with 0.5% SDS treatment for 10 min at 65 °C. SDS was quenched by adding 50 μl 10% Triton X-100 and incubating it for 15 min at 37 °C. The chromosomal DNA in the cell lysate was then digested for 1 h at 37 °C by adding 50 μl 10x NEB buffer 2.1(NEB), 300μl water, and 100U of Sau3AI. Biotin-14-dCTP (TriLINK) was used to label the restriction fragment ends with biotinylated cytosine nucleotides. Labeled DNA was diluted in a 10.0 ml ligation mix containing 100 Weiss units of T4 DNA ligase (Thermo) and ligated overnight at 16 °C. The crosslinking was reversed overnight at 65°C with 200 μg ml-1 proteinase K (Thermo), and DNA was purified using QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's instructions. Purified DNA was sheared to a length of ~400 bp. Biotinylated DNA fragments were pulled down by Dynabeads MyOne Streptavidin C1 beads (Thermo fisher) and prepared for Illumina sequencing by NEBNext Ultra II DNA Library Prep Kit (NEB) as per manufacturer's instructions. The DNA fragments between 400 and 600 bp were purified and paired-end sequenced on the Illumina HiSeq X Ten platform with 150PE mode.