GSM9001296: Wild-type mouse embryonic fibroblasts batch 4, P3; Mus musculus; OTHER
WT and KO MEF samples were in triplicate. Each sample of the MEFs was cultured in 3 10-cm dishes and was synchronized at the G1/S phase as described above. The cells were released into the S phase for 4 hours. They were rinsed twice with PBS and incubated with 500 µg/ml bTMP in 1 ml PBS in the dark on ice for 20 minutes. The cells were carefully covered with parafilm during the incubation to ensure uniform reagent distribution. The plates with parafilm-covered cells were irradiated at a nominal 12 joules/cm2 according to instrumental readings in a UV crosslinker (UVP, Model CL-1000 Ultraviolet Crosslinker, Upland, CA), whose lamps were replaced by 5 8-W T5 365 nm UVA tubes (Coospider-Repta, Jinyun, China). This UV energy level was pre-tested in a dot blot assay (to be described below) to obtain an optimal level of DNA bTMP labeling for MEFs. After the irradiation, the parafilm was removed. The cells were rinsed twice with ice-cold PBS, scraped into 1 ml PBS supplemented with protease inhibitors and pelleted by centrifugation. The cell pellets were treated with 300 µl of lysis buffer (50 mM Tris-HCl, pH 8.1; 10 mM EDTA; 1% SDS) containing protease inhibitors on ice for 10 minutes. The cell lysates were sonicated using a probe-based sonicator (FB120 with CL-18 probe; ThermoFisher) at 25% amplitude, employing 15-second pulses with 15-second intervals for a total of 3.5 minutes. Proteinase K (60 µg) was added to the sonicated sample and incubated at 55°C for 1 hour. Total bTMP-labelled DNA was extracted from the lysate using an equal volume of phenol:chloroform:isoamyl alcohol, precipitated with 0.3M sodium acetate and two volumes of absolute ethanol. The pellet was washed twice with 70% ethanol and resuspended in 50 µl TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). Approximately 45 µg bTMP-labeled DNA extracted from MEFs in 600 l of TE buffer was spiked with 5% (i.e., 2.25 µg) of bTMP-labeled naked DNA from the S2 cells. Fifty µl of prewashed magnetic streptavidin beads (Dynabeads MyOne Streptavidin, Invitrogen, Cat. No. 65001) were washed twice with TE buffer and then added to the DNA mix in a 1.5 ml Eppendorf tube, which was rotated at 4°C overnight. The beads were then sequentially washed with the following buffers: TSE I buffer (20 mM Tris HCl pH8.1, 2 mM EDTA, 150 mM NaCl, 1% Triton, 0.1% SDS) followed by a TE buffer rinse, TSE II (20 mM Tris HCl pH 8.1, 2 mM EDTA, 500 mM NaCl, 1% Triton, 0.1% SDS) followed by a TE buffer rinse, and TSE III (10 mM Tris HCl pH 8.1, 1 mM EDTA, 0.25 M LiCl, 1% NP40, 1% Sodium Deoxycholate) followed by a final TE buffer rinse. Each washing step was 5 minutes, with the Eppendorf tube rotating at room temperature. DNA was then eluted from the beads by 50 µl of 10 mM EDTA/95% formamide at 90 °C for 10 minutes. The eluate was collected and supplemented with 350 µl nuclease-free water. DNA was precipitated by the addition of 40 µl 3M sodium acetate, 1 ml anhydrous ethanol, and 20 µg glycogen in 1 l. The sample was incubated at -70°C overnight, then centrifuged to pellet the enriched single-strand bTMP-labelled DNA. The pellet was subsequently washed with 70% ethanol and resuspended in 15 µl low TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0). Input ssDNA quantity was evaluated using a High Sensitivity DNA Kit (150-1500 pg total). Libraries were prepared using xGenTM ssDNA and low-input Library Prep Kit (IDT) with 11-14 PCR cycles for final library amplification. Library size distribution was assessed on a 2100 bioanalyzer (Agilent Technologies) using a High Sensitivity DNA Kit, and libraries were quantified by qPCR. Equimolar libraries were sequenced in paired-end readcounts (PE100) on a Novaseq system (Illumina) at 35 million readcounts per library.