title: Hi-C of S. cerevisiae arrested  in G1 (alpha factor) depleted in Spt16 and Mcd1 overexpressed Rep 2
design: Aliquots of 1-3 x 109 cells in 150 ml culture medium were fixed in 3% formaldehyde (Sigma-Aldrich, Ref. F8775) and quenched in 0.4 M glycine for 20 min at room temperature and 4C, respectively. Cells were harvested, washed with culture medium and pellets were stored at -80C. Pellets were thawed on ice and resuspended in chilled TE buffer supplemented with protease inhibitors. Cells were lysed using Precellys VK05 Lysing KIT (Ozyme, Ref. 0042) at 6,700 rpm. Lysates were treated with 0.5 % SDS for 20 min at 65C and digested overnight with DpnII (CFinal=500 U/pellet; NEB R0543M) at 37C. Digestions were centrifuged for 20 min at 16,000 g and the pellets suspended in cold water. 5 DpnII overhangs were filled-in and labelled with biotin using 30 M dNTP (dATP, dGTP, dTTP and Biotin-14-dCTP Invitrogen Ref. 19518018). Biotinylated DNA fragments were ligated using T4 DNA ligase (CFinal=250 Weiss U/pellet; Thermo Scientific 10621441) for 4 h at 16C. Cross-link was reverted through an overnight treatment in presence of 250 g/ml proteinase K at 65C. Total DNA was extracted using phenol/chloroform, precipitated and treated with RNAse A 500 g/ml. Non-ligated DNA fragments were removed by T4 DNA polymerase (CFinal=5 U/pellet; NEB M0203L) treatment; whereas ligated fragments were 500 bp sheared using Covaris S220 and pulled-down using Dynabeads Myone Streptavidin C1 (Invitrogen 65001). Resulting libraries were amplified using custom-made primers for paired-end deep-sequencing on NextSeq500 Illumina platform (2x75 bp cycles).
identifiers.namespace: SUB6123371
identifiers.id: Rep2_G1_Gal-Mcd1_Spt16-AID