GSM8781954: BJ NPC WT (Capture Repli-seq); Mus musculus; OTHER
For cell cycle analysis, cells were stained with 80 μg/mL propidium iodide (Invitrogen, P3566) at RT, in the presence of 0.4 mg/mL RNaseA (Roche, #10109169001) for 1h to degrade RNA in the cells. Flow cytometry was performed using an AriaII FACS machine (BD Biosciences) and early and late fractions, and ~250,000 cells were sorted according to the fluorescence strength of propidium iodide. Sorted cells were recovered in a lysis buffer (50 mM Tris-HCl pH=8, 10 mM EDTA, 0.5% SDS, 300 mM NaCl). Genomic DNA samples were extracted through incubation with 0.2 mg/mL Proteinase-K (Thermo Fisher, #EO0492) at 65°C for 2 h, followed by Phenol-Chloroform extraction and EtOH precipitation, and were sonicated into 100-500-bp fragments using a Bioruptor Pico machine (Diagenode), followed by denaturation at 95°C for 5 minutes. BrdU-incorporated DNA was extracted from 250,000 cells and sonicated into 100-500 bp fragments using a Bioruptor Pico (Diagnode). Using NEBNext Ultra II Library Prep Kit for Illumina (New England Biolabs, E7645S) and NEBNext Multiplex Oligo for Illumina (New England Biolabs, E7645S), adaptors was ligated following manufacturer's protocol. DNA was then Phenol-Chloroform purified, denatured (95°C, 5 min), and subjected to BrdU immunoprecipitation. DNA was purified subsequently and was indexed during the amplification stage, using multiplex oligos (New England Biolabs, E7645S) following a manufacturer's method. DNA was then purified with AMPure XP beads (Thermo Fisher, #10136224). The obtained libraries were quantified with Qubit and their quality was assessed with Bioanalyzer 2100 (Agilent Technologies). 4 to12 Repli-seq libraries were pooled in equimolar quantities (1 µg in total) and captured simultaneously, using the Arima Capture Modules (Arima, #A311032, #A311033, #A311034) according to the SureSelect XT HS2 DNA Kits protocol provided by Agilent. Hybridization of Repli-seq libraries with the SureSelect probes was achieved in a thermal cycler with 60 cycles [1 min at 65°C, then 3 sec at 37°C] followed by over-night incubation at 21°C. The hybridized pool of libraries was pulled-down using Streptavidin beads and libraries were then amplified following the manufacturer's instructions, with 12 PCR cycles. Finally, libraries were purified using AMpure XP beads (Beckman Coulter; 1:1 beads:sample ratio), quantified using Qubit dsDNA HS kit (Thermo-Fisher Scientific; Q33230) and their size distribution measured using the TapeStation with the D1000 kit (Agilent). The material was sequenced on an Illumina NextSeq 2000 (paired-end, 100 cycles) at the High-throughput sequencing facility of the I2BC (CNRS, Gif-sur-Yvette).