Rep-ChIC-Seq, FANCD2, WT, Hydroxyurea treated, Replicate 1
Cell were incubated in the presence of 100 mM BrdU for 20 minutes prior to being subjected or not to replication stress using 4mM HU for 1 hour. Following the treatments, 500000 cells were collected and subjected to the ChIC / rep-ChIC procedure as follows. ChIC: Chromatin Immuno-capture or CUT&RUN (Cleavage Under Target & Release Using Nuclease) assay was performed using the ChIC/CUT&RUN Assay Kit from Active Motif (cat#53180) and following the manufacturer instructions. Nuclei were isolated, permeabilized and incubated with the appropriate antibodies (rabbit anti-H3K9me3 [EPR16601] (Ab176916, Abcam), rabbit anti-CTCF antibody (active motif 61311)) according to the manufacturers instructions followed with pAG-MNase binding and activation. Digested fragments were finally solubilized and purified according to the manufacturers. Purified fragments were used to prepare sequencing library using NEB Multiplex Oligos Kit from Illumina. Rep-ChIC: As for ChIC but with the following modification. During the library preparation, after the adapter ligation, DNA was denatured for 5 minutes at 95C and immediately put on ice. The resulting single-stranded DNA was incubated with 0.5 mg anti-BrdU antibody (BD pharmigen cat# 555627, concentration 0,5 mg/mL) for 45 minutes at room temperature. Newly replicated DNA bound to BrdU antibodies was then pulled down using Protein G-Dynabeads. DNA was eluted from the beads using proteinase K and purified using SPRI bead purification. The resulting single-stranded DNA containing sequencing adapter was used as a template to generate indexed Illumina sequencing libraries by PCR.