ATAC-seq on rad50∆5.1/rad50∆5.1 and w1118 CNS
ATAC-seq on rad50∆5.1/rad50∆5.1 and w1118 CNS
ATAC-seq was performed on Wl3 CNS from rad50∆5.1 or w1118 animals in triplicates. 20 CNS for each replicate were dissected on ice in PBS supplemented with 1% BSA. Cells were extracted in 200µL collagenase IV (Gibco ref: 17104019) at 1mg/mL at 37°C for 30'. To enhance dissociation, samples were pipetted 50 times every 10' and 100 times at t=30'. Cells were counted on hemocytometer and 200.000 cells for each reaction were used. Upon dissociation, cells were pellet for 10' at 600g 4°C and nuclei were extracted in nuclei extraction buffer (HEPES pH8.0 20mM, KCl 10mM, Glycerol 20%, triton X-100 0.1%, spermidine trihydrochloride 0.5mM) for 10' on ice. Nuclei were pellet at 1200g for 10' at 4°C.  Tagmentation was performed on extracted nuclei at 37°C for 30' at 800 rpm in tagmentation buffer (Tris-HCl pH 8 33mM, DMF 16%, K-Ac 66mM, MgCl2 10mM, digitonin 0.001%, Tween-20 0.01%, PBS 0.4X) using pre-indexed assembled Tn5 transposomes (Active Motif, ref 53152). DNA was recovered using MinElute purification kit (Qiagen ref: 28004). Libraries were prepared from the whole tagmented DNA using Q5® High-Fidelity 2X Master Mix (NEB) and P5/P7 primers at 1µM final concentration in 50µL total reaction volume and following PCR procedure: 72°C 5'/ 98°C 30''/ (98°C 10'', 63°C 30'', 72°C 1')*12. PCR clean-up was performed using SPRIselect size selection beads (Beckman coulter) with a ratio of 1.2. Library quality was assessed on bioanalyzer using High sensitivity Chips. If adapters remained, those were removed with another size selection step with 1.2 or 1.1 ratio according to amount of contamination.