HiC-Seq of Saccharomyces cerevisiae W303, with mutation Pds5-S81R, stopped in Metaphase (cdc20)
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. These sequences contain home made adaptors that correspond to the first 10 bp of the reads that should be trimmed before alignment.