GSM3656899: HiC_SSY14_ndt80D_2A2_8h; Saccharomyces cerevisiae; Hi-C
For meiotic samples: 5 ml aliquots (20-30 ODs) of S. cerevisiae diploid SK1 cells were fixed with Formaldehyde at 3% final concentration for 20 minutes at 30 °C, 250 rpm, then quenched by incubating with a final concentration of 0.35 M Glycine (2x the volume of Formaldehyde added) for an additional 5 minutes. Cells were washed with water, split into two samples and stored at -80 ̊C ready for library preparation. For mitotic samples: 100ml (50-80 OD) of S. cerevisiae diploid SK1 cells were fixed with Formaldehyde as described above and used for one library.  The Hi-C protocol used was amended from Belton and Dekker (2015, Cold Spring Harb Protoc; doi:10.1101/pdb.prot085209) by ~5-fold reduction in all materials and volumes. Briefly, cells were thawed, washed in spheroplasting buffer (SB, 1 M Sorbitol, 50 mM Tris pH 7.5) and digested for 15-20 min at 35 ˚C with 100 micrograms/ml 100T Zymolyase in SB containing 1% beta-Mercaptoethanol. Cells were washed in restriction enzyme buffer, chromatin was solubilised by adding SDS to 0.1% and incubating at 65 ˚C for 10 minutes. Excess SDS was quenched by addition of Triton X100 to 1%, and chromatin was incubated with 2.07U/ul of DpnII overnight at 37 ˚C. DNA ends were filled-in with nucleotides, substituting dCTP for biotin-14-dCTP using Klenow fragment DNA polymerase I at 37 ˚C for 2 h followed by addition of SDS to 1.5% and incubation at 65 ˚C for 20 minutes to inactivate Klenow and further solubilise the chromatin. The sample volume was diluted 15-fold, crosslinked DNA ends ligated at 16 ˚C for 8 h using 0.024U/ul of T4 DNA ligase, and crosslinks reversed by overnight incubation at 65 ˚C in the presence of proteinase K. DNA was precipitated with ethanol, dissolved in TE and passed through an Amicon 30 kDa column. DNA was further purified by phenol:chloroform:isoamylalcohol extraction and precipitated again before treating with RNaseA at 37 ˚C for 1 h. Biotin was removed from unligated ends by incubation with T4 DNA polymerase at 20˚C for 4 h and at 75 ˚C for 20 minutes for inactivation of the enzyme. DNA was subsequently fragmented using a Covaris M220 (Duty factor 20%, 200 cycles/burst, 350s, 20 ˚C). After purification with with Qiagen MinElute Columns, DNA ends were repaired using T4 DNA polymerase, T4 Polynucleotide Kinase and Klenow fragment DNA polymerase I at 20˚C for 30 minutes. The DNA was purified with Qiagen MinElute Columns and 3' ends were A-tailed with dATP using Klenow Fragment (exo-) at 37˚C for 30 min and then at 65˚C for 20 min to inactivate the enzyme. The Hi-C protocol used was amended from Belton and Dekker (2015, Cold Spring Harb Protoc; doi:10.1101/pdb.prot085209) by ~5-fold reduction in all materials and volumes. Briefly, the end-repaired and A-tailed fragments were fractionated using a Blue Pippin (Sage) and the 100-250bp fraction was used for further processing. Biotinylated fragments were enriched using streptavidin magnetic beads (C1) and NextFlex (Bioo Scientific) barcoded adapters were ligated while the DNA was on the beads. Resulting libraries were minimally amplified by PCR and sequenced using paired-end 42 bp reads on a NextSeq500 (Ilumina; Brighton Genomics).
GEO Accession
GSM3656899