HU_rep2
Homo sapiens
Protocols: Cell culture hTERT immortalized human retinal pigmented epithelial cells (hTERT RPE-1, ATCC ®, CRL-4000™) were grown in T175 flasks at 37 °C with 5 % CO2 with DMEM, high glucose, GlutaMax™, pyruvate (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (10,000 units penicillin and 10 mg streptomycin/mL, Gibco). Cells were passaged with Trypsin-EDTA at 70-80% confluency and used for a maximum of 20 passages. Cells were regularly tested for mycoplasma infection. For Double-Click-seq experiments DMEM containing azidohomoalanine (AHA) and 5-ethynyl-2'-deoxyuridine (EdU) was used. This 100% AHA medium was made as follows: DMEM, high glucose, no glutamine, no methionine, no cysteine (Gibco) was supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin (10,000 units penicillin and 10 mg streptomycin/mL, Gibco), 4 mM GlutaMax™ (Gibco), 0.2 mM L-Cystine 2HCl, 1 mM sodium pyruvate, 0.2 mM azidohomoalanine (AHA) and 20 µM 5-ethynyl-2'-deoxyuridine (EdU). Double-click-seq Incubation and harvest of cells. Five T175 flask were each seeded with approximately 3 x 106 cells per experiment. After overnight incubation, culture medium was removed and replaced by pre-mixed medium containing 0.95 x volume 100% AHA medium (see recipe above) and 0.05 x volume of DMEM (see recipe above). For induction of replication stress either 100 nM curaxin (CBL0137), 150 or 200 µM hydroxyurea (HU), 0.5 µM ATR inhibitor (VE-821, Axon Medchem) or a combination was added to the culture medium. Cells were incubated for 24 h, after which the culture medium was removed and cells were washed twice with ice-cold Phosphate-Buffered Saline (PBS). Cells were harvested with Trypsin-EDTA (Gibco) for 5 minutes at 37 °C. Trypsinization was quenched by the addition of medium and cells were collected by centrifugation at 200 g for 5 minutes. The pellet was resuspended in ice-cold PBS and centrifuged again at 200 g for 5 minutes. The cell pellet was stored at -80 °C until further use. Nuclei isolation. The cell pellet was resuspended in 1 mL of ice-cold PBS containing 0.1% NP-40 supplemented with proteinase inhibitor cocktail (PIC) (cOmplete™, Mini, EDTA-free, Roche). The cell suspension was vortexed for five seconds at maximum speed. The lysed cell suspension was centrifuged for 10 seconds at 9000 g. The supernatant was discarded and the nuclei were resuspended in 1 mL of ice-cold PBS containing 0.1% NP-40 supplemented with PIC. The washed nuclei were centrifuged for 10 seconds at 9000 g and the supernatant was discarded. First click reaction. The nuclei were resuspended in 250 µL nuclei buffer (320 mM sucrose, 5 mM MgCl2, 10 mM HEPES at pH 7.4) supplemented with PIC and 10 µL 100 mM (in methanol) biotin-PEG4-alkyne (Merck) was added. Next, a pre-mixed solution of 5 µL 50 mM CuSO4 with 5 µL 250 mM THPTA (Merck) was added, followed by 10 µL of a freshly prepared solution of 100 mM sodium ascorbate. The nuclei were placed in an end-over-end rotator at room temperature for 30 minutes. After that, the click reaction was repeated as described above using fresh reagents. Nuclei were pelleted for 10 seconds at 9000 g (to remove excess biotin, which would interfere with streptavidin bead pull-down later on) and the supernatant was discarded. MNAse digestion. Nuclei were resuspended in 100 µL of MNAse buffer (50 mM Tris.HCl, 5 mM CaCl2, pH 7.9, supplemented with 100 µg/mL BSA) and warmed to 37 °C in a water bath. Then, 60 U micrococcal nuclease (New England Biolabs, MNase, 1U/µL in MNAse buffer) was added and cells were incubated at 37 °C for 5 minutes. MNase reactions were stopped by adding EDTA to a final concentration of 5 mM and by cooling on ice. Next, 100 µL PBS supplemented with 0.1% BSA and 0.01% Tween was added and the nuclei were pelleted by centrifugation for 10 seconds at 9000 g. Streptavidin enrichment of labeled nucleosomes. The supernatant (containing the nucleosomes) was added to 30 µL streptavidin magnetic beads (Dynabeads™ MyOne™ Streptavidin C1, Thermo Scientific, pre-washed three times with PBS according to the instruction manual) and incubated at room temperature for 1 hour in an end-to-end rotator. The supernatant was removed by placing it on a magnet for 3-4 minutes. The beads were washed five times by resuspending them in 200 µL PBS supplemented with 0.1% BSA and 0.01% Tween and placing them back on the magnet. After washing, the DNA bound to the captured nucleosomes was released. This was done by resuspending the beads in 100 µL PBS supplemented with 1 mg/mL proteinase K and incubating the suspension for 30 minutes at 50 °C (pipet up and down after 15 minutes to ensure a homogenous mixture). Then, 100 µL 5 M NaCl was added and the mixture was left rotating for 15 minutes at room temperature. Finally, 360 µL (1.8 x volume of sample) Ampure beads (Beckman) was added directly to the suspension to purify the released DNA according to the protocol of the manufacturer. The DNA was eluted in 80 µL water. Second click reaction. 1 µL 100 mM (in methanol) biotin-PEG4-azide was added to the eluted DNA, followed by a pre-mixed solution of 5 µL 50 mM CuSO4 with 5 µL 250 mM THPTA and 10 µL of a freshly prepared solution of 100 mM sodium ascorbate. The sample was placed in an end-over-end rotator at room temperature for 45 minutes. To discard DNA fragments larger than approximately 200 bp 70 µL (0.7 x volume of sample) Ampure beads were added and after 5 minutes incubation at room temperature the beads were discarded and the supernatant was transferred to a new tube. Then 180 µL (1.8 x original sample volume) Ampure beads were added and purification was completed according to the manufacturer's protocol. The DNA was eluted in 55.5 µL water. End prep and adaptor ligation. End repair, 5' phosphorylation, dA-tailing and adaptor ligation was performed using the NEBNext® Ultra™ DNA library prep kit for Illumina®. After adaptor ligation the sample was added to 25 µL streptavidin magnetic beads (Dynabeads™ MyOne™ Streptavidin T1, Thermo, pre-washed three times with B&W buffer (5 mM Tris HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl ) according to the instruction manual). Isolation of replicated double stranded DNA fragments. After 15 minutes incubation at room temperature in an end-over-end rotator, beads were washed four times with 200 μl 1x B&WT buffer (5 mM Tris HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05 % Tween 20) and once with 2 x B&WT buffer. Isolation of replicated parental strands. The parental strands were then eluted from the beads by incubation with 100 μl alkaline buffer (0.1 M NaOH, 0,05 % Tween 20) for 1 minute. Elution of the parental strand was repeated twice for a total of three times and the pH of the combined alkaline supernatants was neutralized by adding acetic acid to a final concentration of 0.1 M and 2 mM EDTA pH 8.0. The sample was diluted to 1 mL with water and concentrated to approximately 20 uL with a Vivaspin® 2 5K according to the manufacturer's instruction (15 minutes centrifuging at 4000 g in a swing bucket). The DNA was recovered by performing a reverse spin at 3000 g for 2 minutes. Library amplification and sequencing. Libraries were amplified using the NEBNext® Ultra™ DNA library prep kit for Illumina® using NEBNext® multiplex oligos for Illumina® (index primer set 1 or 2) with 13 cycles of PCR. Purification was done as described in the manual (with 0.9 x Ampure beads) and eluted in 30 μl water. Libraries were quantified with Qubit™ dsDNA high sensitivity assay kit (Thermo) and further analyzed with the high sensitivity D1000 ScreenTape system (Agilent).