Transcriptional repression accounts for RNA:DNA hybrids accumulation at DNA Double Strand breaks
Transcriptional repression accounts for RNA:DNA hybrids accumulation at DNA Double Strand breaks
ENDseq: Briefly, 15x10^6 DIvA cells per condition were collected by trypsination. DIvA (AsiSI-ER-U20S) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics, 10% FCS (InVitrogenInvitrogen) with 1 µg/mL puromycin (DIvA cells) at 37°C under a humidified atmosphere with 5% CO2. AsiSI dependent-DSB were induced by the addition of 300 nM 4-OHT (Sigma, H7904) into the medium for 4 h or 24 h.siRNA transfections were performed using 4D-Nucleofector from Lonza either with the SE-small kit for 1 million cells or SE-large kit for 5-20 million of cells, according to the manufacturer's instruction. Cells were collected 48 h after siRNA transfection ENDseq:cells were embedded into two or three 0.75% agarose plugs (BioRad CHEF Mammalian genomic DNA Plug Kit). Agarose plugs were then transferred at 4°C for 1 h to allow polymerization. Plugs were then transferred into 15 mL tubes in which they were subjected to proteinase K treatment for 1 h at 50°C followed by 7 h at 37°C. All rinses and washes were performed in 15 mL. Plugs were then rinsed three times plug wash buffer (50 mM EDTA pH 8 and 10 mM Tris HCl pH 8) followed by two rinses with TE buffer (10 mM Tris HCl pH8 and 1 mM EDTA) and two 15 min washes with TE buffer on a rotating wheel. Plugs were next treated with RNAse A for 1 h at 37°C in TE buffer. Plugs were subsequently rinsed three times in plug wash buffer followed by four 15 min washes in plug wash buffer. Plugs were stored over the weekend at 4 °C in plug wash buffer. Next, plugs were rinsed twice in EB buffer (10 mM Tris HCl pH 8) followed by four 15 min washes in EB buffer on a rotating wheel. For the spike in DNA, 100 ng of the AsiSI digested “E171” plasmid (that contains a single AsiSI site) was also embedded into agarose plugs ENDseq:Plugs were subjected exonuclease VII digestion (NEB) for 1 h at 37 °C followed by two washes in 1x NEBuffer 4 before digestion with exonuclease T for 1 h at 25°C. Plugs were then washed three times for 15 min in EB buffer on a rotating wheel followed by two washed in NEBNext dA-Tailing buffer. Plugs were then treated Klenow fragment (3'-5' exo-) for 1 h at 37°C. After removal of the enzymatic mix, plugs were washed in 1X NEBuffer 2 for 15 min on a rotating wheel. After removal of the wash, plugs were subjected to ligation with the Quick Ligase (NEB) in the presence of 10 μM of pre-annealed END-seq adapter 1 (Wong et al, 2020) for 1 h at 25°C. Plugs were washed twice in plug wash buffer before being decanted overnight in plug wash buffer. The next day, plugs were rinsed twice in TE buffer followed by four 15 min washes in TE buffer on a rotating wheel. Individual plugs were then melted for 2 min at 70°C followed by 5 min at 43°C. Beta-agarose was added and incubated for 45 min at 43°C. The entire DNA solution was then transferred onto a 0.1 μm dialysis membrane pre-equilibrated in TE buffer during 1 h at room temperature. DNA was next subjected to proteinase K digestion in the presence of SDS for 15 min at 50°C. The DNA was then transferred into Covaris S220 microtubes (PN520045) for DNA fragmentation (Covaris S220, 175W peak incident power, 10% Duty factor, 200 cycles per burst for 240 sec). Fragmented DNA was precipitated using ethanol and glycogen. Samples were resuspended into TE, and centrifuged again to remove any residual white pellet that would not correspond to DNA being precipitated. DNA is quantified and stored at -20°C. The next day, only 1/1000 of the “E171” plasmid DNA added to each condition for a spike in control. The fragmented DNA was the precipitated in 1X WB (5 mM Tris HCl pH 8, 0.5 mM EDTA, 1 M NaCl) using Dynabeads MyOne magentic C1 beads (ThermoFisher) for 30 min at room temperature. Beads were then washed three times with 1X WB, followed by two washes with EB buffer and once with 1x NEB T4 DNA ligase buffer. Samples were then incubated with an end repair a mixture of the large Klenow fragement, T4 PolI and T4 PNK for 30 min at 24°C. Beads were washed once with 1X WB, twice with EB buffer and once with 1X NEBNext dA-tailing buffer. After that, beads were incubated with the dA-tailing reaction mix containing the Klenow fragment (3'-5' exo-) for 30 min at 37°C. After removal of the dA-tailing reaction, samples were subjected to ligation with the Quick Ligase (NEB) in the presence of 0.5 μM of END-seq adapter 2 (Wong et al,2020) for 20 min at 25°C. The ligation reaction was inactivated with EDTA and beads were washed three times with 1X WB and three times with EB. The USER enzyme (NEB) was added to samples for 30 min at 37°C. 20 cycles of PCR reaction was achieved with NEBNext multiplex oligos for Illumina (E7335S, New England Biolabs) and PCR products were purified with SPRIselect or Ampure XP beads (Beckman Coulter) using first a size selection with a 0.6X beads-to-sample ratio followed by two consecutive purifications with a 0.9X ratio.