RNAseq of RAD50∆5.1 null mutant and WT W1118 larval stage 3 CNS
RNAseq of RAD50∆5.1 null mutant and WT W1118 larval stage 3 CNS
L3 CNS (10 per replicate) were quickly dissected on ice in PBS, put directly in 150µl TRIzol™ (Invitrogen, ref 15596026) once a full sample was collected and stored on ice.  CNS were lysed with 10 strokes of microtube pestle (pestle was rinsed with another 150µl TRIzol™), vortex at full speed for 1min and incubate 10 min at RT for full lysis. 1/5 V of Chloroform was added and samples were vortexed 1min at full speed, incubated 5 min at RT and centrifuged at 16000g for 30 min at 4°C. Supernatant was collected, and RNA was extracted with 1V of isopropanol and glycogen (3 µg/mL final concentration), incubate at -20°C for 2h and centrifuge at 16000g for 30 min at 4°C. Pellets were washed twice in ethanol 75%, 15 min at 16000g 4°C, allowed to dry quickly, resuspend in 30µl H2O, treated with 25U DNAse I (Thermo Scientific™ 89836) for 5 min in 50µL total volume at RT. RNA was extracted a second time in 500µL TRIzol™ like previously described. End washing steps were performed 3 times. Total RNA was resuspended in 15µL H20, 2µL were kept for downstream analysis and samples were immediately stored at -80°C till processing. RNA integrity was analyzed on Agilent RNA Chip on bioanalyzer 2100.  Library preparation was performed at the GenomEast platform (IGBMC) using using the Illumina® Stranded mRNA Prep.