Repli, Early, Untreated, Replicate 1
Exponentially growing MRC5 cells were pulse-labelled with 100 M BrdU for20minutes. Immediately after the BrdU pulse cells were collected by trypsinization and fixedin 70% ethanol and incubated overnight at 4C in the presence of 15 g/mL Hoechst 33342.Cells were re-suspended in 1xPBS and sorted in 2 fractions (100,000 cells per fraction): early(E) s-phase cells and late (L) s-phase cells. Sorted cells were incubated overnight in lysis buffer(50mM Tris-HCl pH 8, 10mM EDTA, 0.1% SDS, 50g/mL RNAse A, 100g/mL ProteinaseK). DNA was purified from the lysates by phenol extraction. Purified DNA was sonicatedusing a Covaris sonicator, and fragments were size-selected to 200300 bp using SPRI beads.Prior to pull-downs, size-selected DNA was end-repaired, A-tailed, and ligated to paired-endIllumina adapters as in Rep-Hi-C. Adapter-ligated DNA was first used to enrich replicatingregions by capturing BrdU-labelled fragments, as in Rep-Hi-C. The resulting single-strandedDNA after BrdU capture was PCR-amplified to the desired number of cycles and sequenced on an Illumina NovaSeq 6000 platform.
SUB15914284
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