title: Fork_deg_seq, G9ai, phIAA minus, Hydroxyurea treated
design: 10-15 million cells were incubated in the presence of 100 mM BrdU for 40 minutes prior to being subjected replication stress using 4mM HU for 5 or 8 hours. 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. 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 treated with 0.1% SDS for 10 mins at 65C and quenched with 1% Triton X-100 to permeabilized nuclei. Biotinylated nucleotide was incorporated at degradation sites by a gap filling reaction, followed by ligation, de-crosslinking and DNA purification as explained above in the rep-HiC procedure. The DNA was then sonicated using a Covaris sonicator and SPRi beads-based size selection (200-300bp) of DNA fragments, desired for library preparation. Before pulldowns, the size selected DNA was repaired, A-tailing and PE Illumina adapter ligation similar to rep-HiC. Adapter ligated DNA was used as input to enrich all the replicating sites by capturing BrdU-labelled fragments as in rep-HiC. The ssDNA after BrdU capture was thereafter used to capture all degradation sites by using streptavidin beads to pulldown biotin incorporated at ligation sites. The bead-bound DNA was then PCR amplified to the desired cycle and sequenced using the Illumina NovaSeq 6000 sequencing platform.
library_name: 2_S10