GSM7666983: synIV_yWZ536; Saccharomyces cerevisiae; Hi-C
Crosslinked cells were harvested and suspended in 10 mL spheroplast buffer (1M sorbitol, 5 mM DTT, 250 U zymolyase 100T (US Biological, Z1004)) for 40 min incubation at 30°C. Spheroplasts were washed with 10 mL of 1M sorbitol once and resuspended in 2 mL of 0.5% SDS at 65°C for 20 min. MboI (NEB, R0147) was used for overnight digestion of all the genomic DNA from fixed yeast cells at 37°C. The digestion product was centrifuged at 18000xg for 20 min and the pellet was subsequently suspended in 200 mL cold water. DNA sticky ends were filled with biotin-14-dCTP (Invitrogen, 19518018) by Klenow enzyme (NEB, M0210L) at 37°C for 80 min. Biotinylated DNA was ligated with T4 ligase (Thermo Scientific, EL0014) at room temperature for 2 hours. Ligation product was reverse cross-linked by 0.5 mg/mL proteinase K (Thermo Scientific, EO0492) in 25 mM EDTA buffer at 65°C for 4 hours. The reverse crosslinked sample was purified using the large fragment DNA recovery kit (Zymo Research, D4046). Purified Hi-C library was used as input material for Illumina sequencing library prep kit (NEB, E7805).