GSM7814738: clone3D1_mit_exp5; Homo sapiens; OTHER
EdU-labelled DNA isolation and sequencing were performed as described in Mailler, Padayachy & Halazonetis, 2021, and Macheret & Halazonetis, 2019. Briefly, fixed cells were permeabilized in PBS-0.2% Triton X (PanReac, AppliChem, Cat. No. A1388,2500) and resuspended in click-it reaction mix (85.5mM Tris pH8, 4mM CuSO4, 100mM Sodium Ascorbate and 50µM biotin-azide, Jena Biosciences, Cat. No. CLK-A2112-10) for 30 minutes for biotinilation of EdU-labelled DNA. Each sample was then incubated over-night in a solution of 10mM Tris pH 8, 10mM EDTA, 0.5% SDS and proteinase K (Roche, Cat. No. 03115844001) 0.2mg/mL. The next day, proteins and other contaminants were eliminated by phenol-chlorophorm extraction, and purified DNA was precipitated over night at -20°C by adding 0.2M NaCl and two volumes of ethanol to the solution. DNA pellet was washed in 70% ethanol, resuspended in 1xTE (10mM Tris pH 8, 1mM EDTA) and quantified by Qubit ® dsDNA BR assay kit (Invitrogen, Cat. No. Q32853) using a Qubit ® 2.0 Fluorometer (Invitrogen, Cat. No. Q32866). 10μg of DNA from each samples were sonicated by Bioruptor ® Pico sonication device (Diagenode, cat. no. B01060003) to get 100-500bp fragments (6-8 cycles, 15 seconds on, 90 seconds off). EdU-biotin-labelled DNA was isolated by using Dynabeads™ MyOne™ Streptavidin C1 (Invitrogen, Cat. No. 65002) and eluted in Tris 10mM pH8 additioned with 2% β-mercaptoethanol. TruSeq ChIP Sample Prep Kit (Illumina, Cat. No. IP-202-1012)