title: GSM8718541: HME1_Myc_EdU_HUseq_Rep2; Homo sapiens; OTHER
library_name: GSM8718541
library_construction_protocol: Stable Ctrl-KO, MSL3-KO and Myc overexpressing HME1 cells were seeded in T150 flasks. When 50% confluency was reached, cells were treated with 2μM Palbociclib (Universal Biologicals, S116) for 20 hours to synchronize them in G1/S. Cells were washed twice with medium and treated with 10mM Hydroxyurea (Merck Life Science, H8627) for 7 hours to stop them at the entry of S phase. After two washes with medium, cells were released in medium containing 25μM EdU (Universal Biologicals, 10540) for 90 minutes. Cells were collected and fixed in 90% MetOH. EdU-labelled DNA was isolated as previously described[5]. Briefly, cells were permeabilized in 0.2% Triton-X100 in PBS for 30 minutes and EdU was detected using a biotin-azide click reaction 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, cells were 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). For HighResEdU-HUseq sample, stable Ctrl-KO HME1 cells were seeded in T150 flasks. When 50% confluency was reached, cells were treated with 100ng/ml Nocodazole (Merck Life Science, SML1665) for 8 hours. Cells in mitosis were detached by shake-off in the same culture medium, pelleted, washed once with PBS and plated in medium containing 25μM EdU (Universal Biologicals, 10540) and 10mM Hydroxyurea (Merck Life Science, H8627). After 16 hours, cells were collected and fixed in 90% MetOH. EdU-labelled DNA isolation and library preparation were carried out as described for EdU-HUseq. DNA libraries were prepared using the NEBNext UltraII DNA Library Prep Kit for Illumina (New England Biolabs, E7645L).