title: GSM8718522: REPLIseq_WT_late_R1; Homo sapiens; OTHER
library_construction_protocol: Stable Ctrl-KO and MSL3-KO HME1 cells were seeded in T150 flasks. While growing in exponential phase, asynchronous cells were pulsed with 25μM EdU (Universal Biologicals, 10540) for 30 minutes. After fixation with 90% MetOH and permeabilization, Biotin Click it reaction was performed by incubating cells in click-it reaction cocktail (100 mM TrisHCl pH 8, 4mM CuSO4, 100mM sodium-L-ascorbate, 50µM Biotin Azide - Thermo Fisher Scientific, B10184) for 30 minutes. After washes, DNA was stained using PI (propidium iodide). Two population of cells, corresponding to early and late S phase cells were sorted using Beckman Coulter - MoFlo™ XDP. Cells were pelleted and incubated in lysis buffer (10 mM Tris-HCl, pH 8, 10 mM EDTA, 0.5% SDS, 0.2 mg/ml Proteinase K) for 3 hours at 50ºC. DNA was purified using phenol/chloroform/isoamyl alcohol, resuspended in TE and sonicated using Bioruptor Pico. EdU-labelled DNA was captured using Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific, 65001), washed and eluted from beads using 2% β-mercaptoethanol. DNA was purified with solid-phase reversible immobilization (SPRI) beads (Agencourt AMPure XP, Beckman Coulter), and then quantified with QuantiFluor (Promega, E2670). DNA libraries were prepared using the NEBNext UltraII DNA Library Prep Kit for Illumina (New England Biolabs, E7645L).