Rep-ChIC-Seq, H3k9me3, WT, Hydroxyurea treated, Replicate 2
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.