NextSeq 500 sequencing; DNA double-strand breaks and 3D chromatin conformation (Hi-C, 4C-seq, ChIP-seq)
DNA double-strand breaks and 3D chromatin conformation (Hi-C, 4C-seq, ChIP-seq)
yeast_PDS5AID_NoIAA_gal0min_s
For the ChIP experiment in yeast, individual colonies of yFZ014 and yFZ016 were grown in YEP + 3% lactic acid (YEP-Lac) until log phase growth with a final cell concentration between 5×106 cells/ml and 8×106 cells/ml. Degradation of Pds5::9myc-AID in yFZ016 was induced by addition of auxin (indole-3-acetic acid (IAA) Sigma Aldrich # I3750) at a final concentration of 1 mM and confirmed by Western blotting.  HO expression is induced by adding 2% galactose during 75min (final concentration) to the medium. For chromatin immunopreciptiation, 45 ml of culture was fixed and cross-linked with 1% formaldehyde for 10 min, after which 2.5 ml of 2.5 M glycine was added for 5 min to quench the reaction. Cells were pelleted and washed 3 times with 4°C TBS. Yeast cell walls were disrupted by beating the cells with 425-600 µm glass beads for 1 h in lysis buffer at 4°C. The lysate was sonicated for 2 min to obtain chromatin fragments of ~500 bp in length. Debris was then pelleted and discarded, and equal volume of lysate was immunoprecipitated using γ-H2A antibody for 1 h at 4°C, followed by addition of Protein-A agarose beads (Sigma-Aldrich #1719408001) for 1 h at 4°C. The immunoprecipitate was then washed twice in 140 mM NaCl lysis buffer, once with 0.5 M NaCl lysis buffer, once with 0.25 M LiCl wash buffer and once with TE. Crosslinking was reversed at 65°C overnight followed by proteinase K and glycogen addition for 2 h. Protein and nucleic acids were separated by phenol extraction. LiCl was added to a final concentration of 400 mM LiCl. DNA was precipitated using 99.5% EtOH. A second precipitation step was carried out using 75% EtOH and the DNA resuspended in TE.  For ChIP: Sequencing libraries were prepared from IP of SCC1 and pATM(S1981) and input samples using the NEBNext® Ultra™ II Library Prep Kit (NEB)for Illumina. MDC1 libraries were prepared at EMBL Genomics core facilities (Heidelberg, Germany) using NEBNext ChIP-Seq Library Prep kit (New England Biolabs). Sequencing libraries were prepared and sequenced as for ChIP-seq in human cells.