title: NextSeq 500 sequencing; Senataxin resolves R-loops forming at DNA double strand breaks to prevent translocations
design: Senataxin resolves R-loops forming at DNA double strand breaks to prevent translocations
library_construction_protocol: 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.  For AsiSI-dependent DSB induction, cells were treated with 300 nM 4OHT (Sigma; H7904) for 4h ChIP: Formaldehyde was added to the culture medium at a final concentration of 1% and crosslinking was allowed to proceed for 15 min at room temperature. Reaction was stopped by adding glycine (0.125M final concentration) and incubation for 5 minutes at room temperature. Cells were washed twice with PBS and harvested by scraping. Cell lysis was performed by incubation in cell lysis buffer (5 mM Pipes pH 8, 85 mM KCl, 0.5% IGEPAL® CA-630) followed by Dounce homogenization. Nuclei were harvested by centrifugation and incubated in nuclear lysis buffer: (50 mM Tris pH 8.1, 10 mM EDTA, 1% SDS) and sonicated 10 times for 10 s at a power setting of 5 and 50% duty cycle (Branson Sonifier 250). Samples were diluted 10 times in ChIP dilution buffer (16.7 mM Tris pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X‐100, 0.01% SDS) and precleared using a mixture of protein A-agarose (Pierce) and protein G-sepharose (Sigma) previously blocked with BSA (Sigma). Precleared samples (200 μg) were incubated overnight at 4°C with indicated antibodies. Immune complexes were recovered by incubation with blocked protein A/protein G beads for 2 h at 4°C on a rotating wheel. Beads were washed once in dialysis buffer (50 mM Tris pH 8, 2 mM EDTA, 0.2% Sarkosyl), five times in wash buffer (100 mM Tris pH 8.8, 500 mM LiCl, 1%  IGEPAL® CA-630, 1% NaDoc) and twice with TE (10mM Tris-HCl, pH 8, 1mM EDTA). Beads were resuspended in TE supplemented with 50µg/ml DNase-free RNase A (Abcam) and incubated for 30 minutes at 37°C. SDS (final concentration 0.5%) was added and crosslink was reversed by overnight incubation at 70°C. Following a 2 hours treatment with 200 µg/ml proteinase K (Roche) at 45°C, DNA was purified by phenol/chloroform extraction and recovered by ethanol precipitation in presence of 5 µg glycogen (Invitrogen). DNA pellet was resuspended in water. Prior to next-generation sequencing library preparation, samples from multiples ChIP experiments were pooled and sonicated for 5 cycles (30 seconds on, 30 seconds off, high setting) on a Bioruptor (Diagenode) then concentrated with a vacuum concentrator (Eppendorf). For SETX ChIP, 100 µg of chromatin was immunoprecipitated by using 2 µg of anti-SETX (Novus Biologicals, NB100-57542). For RNA Pol II ChIP, 25 µg of chromatin was immunoprecipitated with 75 µl of anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) (Chromotek 3E10) or with 2µg of the anti-total RNA PolII (Bethyl Laboratories A304-405A).  DRIP-Seq: DIvA cells were treated with 300 nM 4OHT for 4h, trypsinized, pelleted at low speed and washed with DPBS (Life technology). Total nucleic acids were extracted with 0,5% SDS /Proteinase K (Thermo Fisher Scientific, Waltham, MA) treatment at 37°C overnight and recovered by phenol-chloroform extraction and ethanol precipitation. DNA was digested by a restriction enzyme cocktail (20 units each of EcoRI, HindIII, BsrGI, XbaI) (New England Biolabs) in 1× NEBuffer 2, with or without RNase H treatment, overnight at 37°C. Fragmented DNA was cleaned by phenol-chloroform extraction and ethanol precipitation followed by two washes with 70% ethanol. Air-dried pellets were resuspended in 10 mM Tris-HCl pH 7.5, 1 mM EDTA (TE). 4 µg of digests was diluted in 450 µL of TE, and 10 µL was reserved as input for qPCR. 50 µL of 10× IP buffer was added (final buffer concentration of 10 mM sodium phosphate, 140 mM sodium chloride, 0.05% Triton X-100) and 10 µL of S9.6 antibody (1 mg/ml, kind gift from F. Chedin, UC Davis). Samples were incubated with the antibody at 4°C for 2 hours on a wheel. 50 µL of Protein A/G Agarose (Pierce), previously washed twice with 700 µL of 1× IP buffer for 5 minutes at room temperature, were added and samples were incubated for 2h at 4°C on a wheel. Each DRIP was then washed three times with 700 µL 1× IP buffer for 10 minutes at room temperature. After the final wash, beads were resuspended in 250 µL of 1× IP buffer and incubated with 60 units of Proteinase K for 45 minutes at 55°C. Digested DRIP samples were then cleaned with phenol-chloroform extraction and ethanol precipitation. Air-dried DRIP pellets were resuspended in 45 µL of 10 mM Tris-HCl pH 8.  For DRIP-seq, samples from 3 DRIP experiments were pooled and sonicated to an average size of 300bp using a Bioruptor (Diagenode) for 20 cycles of 30 s On, 30 s Off, High setting. Immunoprecipitated DNA was subjected to library preparation at  EMBL Genomics core facilities (Heidelberg, Germany).  RNA-Seq: For RNA-seq, DIvA cells (transfected with the control siRNA) were lysed using TRI reagent (SIGMA) and spiked-in with ERCC RNA Spike-In Mix (Thermo Fisher Scientific). Total RNA were recovered by chloroform extraction followed by isopropanol precipitation. Samples were treated with RQ1 RNase-free DNase (Promega) for 1 hour at 37 C and purified by phenol/chloroform extraction followed by ethanol precipitation. Ribosomal RNA depletion and RNA-Seq library preparation were performed at EMBL Genomics core facilities (Heidelberg, Germany) using TruSeq Stranded Total RNA (Illumina). ChIP-seq and DRIP-seq library prepartion was perfomed EMBL Genomics core facilities (Heidelberg, Germany) using NEBNext ChIP-Seq Library Prep kit (New England Biolabs). RNA-seq library preparation were performed at EMBL Genomics core facilities (Heidelberg, Germany) using TruSeq Stranded Total RNA (Illumina).
attributes.tag: Experimental Factor: stimulus
attributes.value: none
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: input DNA
attributes.tag: Experimental Factor: protocol
attributes.value: ChIP-seq