HiC-Seq of Saccharomyces cerevisiae W303, Pds5-degron, stopped in Metaphase (cdc20) bank 2
Aliquots of 1-3 x 109 cells in 150 ml YPD/synthetic medium were fixed in 3% formaldehyde (Sigma) for 20 min at room temperature and quenched with 25 ml glycine 2.5 M for 20 min at 4C. Cross-linked cells were recovered through centrifugation, washed with YPD and a 150 mg pellet was stored at -80C. The pellet was thawed on ice and cells were disrupted using a Precellys apparatus (Bertin Instruments).The spheroplasts were treated with 3% SDS for 20 min at 65C and the lysate was digested overnight with DpnII (CFinal=450 U/pellet; NEB) at 37C. The digestion was centrifuged for 20 min at 18000 g, the supernatant discarded and the pellet suspended in 400 L of water. The 5 overhangs from DpnII digestion were filled in using dNTP 30 M (biotin-14-dCTP, dATP, dGTP and dTTP; Invitrogen), at 37C for 45 min. The biotinylated DNA fragments were ligated by T4 DNA ligase (CFinal=250 Weiss U/pellet; Thermo Scientific) for 4 h at 16C. DNA purification was achieved through an overnight incubation at 65C in presence of proteinase K 250 g/ml and EDTA 6.2 mM, followed by a phenol/chloroform extraction on the precipitated DNA and an RNAse A DNase-free 500 g/ml treatment. Hi-C DNA libraries were 300 bp sheared, using CovarisS220apparatus, and the biotin-labeled fragments were selectively captured by Dynabeads MyoneStreptavidin C1 (Invitrogen). The resulting libraries were used as template for the lluminaamplification by PE-PCR primers and paired-end sequenced on the NextSeq500 or HiSeq 2000 Illumina platforms.