Chromosome Conformation Capture of Saccharomyces cerevisiae genome in G0 phase (strain: BY4741).
Chromosome Conformation Capture of Saccharomyces cerevisiae genome in G0 phase.     The cells were cross-linked  for 20 minutes with fresh formaldehyde (3% final concentration), pooled as aliquots of  3 x 109 cells, and stored at -80°C until use. Aliquots were thawed on ice and  resuspended in 6 ml 1X DpnII buffer (NEB). The cells were then split into four tubes  and lysed using a Precellys grinder (3 cycles: 6500 rpm – 30 sec ON / 60 sec OFF)  and VK05 beads. The cells were incubated for 3 hours with 50 units of restriction  enzyme under agitation (NEB, DpnII). The digestion mix was then diluted into ligation  buffer and a ligation was performed at 16°C for 4 hours followed by a decrosslinking  step consisting in an overnight incubation at 65°C in presence of 250μg/ml  proteinase K in 6.2mM EDTA. DNA was then precipitated, resuspended in TE buffer  and treated with RNAse.  The resulting 3C libraries were sheared and processed into Illumina libraries using  custom-made versions of the Illumina PE adapters (Paired-End DNA sample Prep Kit  – Illumina – PE-930-1001). Fragments of sizes between 400 and 800 bp were  purified using a PippinPrep apparatus (SAGE Science), PCR amplified, and paired-  end (PE) sequenced on an Illumina platform (HiSeq2000 or NextSeq).
Institut Pasteur
G0_BY4741_bk149_BC176_end1.pcrfree_L4_HiSeq_sept2013