title: Rep-Hi-C, DpnII, Untreated, Replicate 1
design: 60-70 million cells were grown in 15 cm dishes with a confluency of 70% and were pulse  labelled with 100 mM BrdU for 20 minutes after which were incubated or not with 4mM HU  for 1 hour to induce replication stress. Cells were then cross-linked using 1% formaldehyde for  10 minutes at room temperature, quenched using 300mM glycine for 5 minutes at room  temperature followed by a 15 minutes incubation at 4C. Fixed cells were collected by  scraping, snap frozen using liquid nitrogen and stored at -80C until further use. Crosslinked  pellets were dounced and treated with a pre-cooled permeabilization buffer (10mM Tris-HCl  ph 8.0, 10mM NaCl, 0.4% Igepal CA-630 and protease inhibitor cocktail) for 1 hour. Cells  were then washed with 1X NEbuffer 2.1 and treated with 0.1% SDS for 10 mins at 65C and  quenched with 1% Triton X-100 to permeabilized nuclei. The open chromatin was then  digested by 400U DpnII restriction enzyme reaction in 1X NEbuffer 2.1, overnight at 37C.  Biotinylated nucleotide was incorporated at interacting sites by a gap-filling reaction at 23C  for 4 hours followed by blunt end ligation reaction at 16C for 4 hours. The ligated DNA was  then de-crosslinked using standard proteinase K procedure overnight. Protein-free DNA was  purified using phenol and chloroform, ethanol precipitation and amicon columns to remove  excess salt and concentrate the DNA (adapted from (20).  After DNA purification, the unligated/dangling fragments with unwanted biotinylated  nucleotides were removed before sonicating using a covaris sonicator and SPRi beads-based  size selection (200-300bp) of DNA fragments, desired for library preparation.  Prior to  pulldowns, the size selected DNA was repaired using T4 DNA polymerase, T4 PNK, Klenow  DNA Polymerase and dNTPs and purified using SPRi beads. The repaired DNA was used for  A-tailing and followed with adapter ligation. Adapter ligated DNA was divided into two  fractions. The first fraction was used to generate HiC libraries and was directly subjected to  the capture of biotinylated DNA fragment using magnetic beads coated with streptavidin  (MyOne streptavidin beads Invitrogen). DNA bound to the beads was finally used as a PCR  template to generate indexed HiC libraries for Illumina sequencing.  The second fraction was used to perform the rep-HiC procedure as follow. Adapter ligated  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) per mg DNA 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 then incubated with  streptavidin coated beads (MyOne streptavidin beads Invitrogen). DNA bound to the beads  was finally used as a PCR template to generate indexed rep-HiC libraries for illumina  sequencing using the Illumina NovaSeq 6000 sequencing platform.
library_name: rep_HiC_UT_DpnII_Bio_2_part_1_lane_2
identifiers.namespace: SUB15094211
identifiers.id: rep_HiC_UT_DpnII_Bio_2_part_1_lane_2