ATAC-seq on sorted neurons from staged E16 or L3 neurons
ATAC-seq on sorted neurons from staged E16 or L3 neurons
Brains : 70 brains dissected in PBS 250µL, pellet 3' at 4°C 600g, resuspent in 200µL 1mg/mL collagenase D 1h at 700rpm 37°C. Isolated cells washed once in PBS at 600g 10' 4°C and resuspend in 300µL PBS and pass through FACS filter for sorting. 10 oregon brains were used as negative control for FACS. E16 embryos are collected, washed, dechorionated 1min in bleach, washed extensively and crushed using a dounce tissue grinder, first using the loose pestle than the tight one. Cells are FACSed using 100µm nozzle. Strain used : elav-nRFP  Nuclei isolation : Add 200µL ice cold nuclear extraction buffer (HEPES-KOH pH 7.9 20mM, KCl 10mM, Spermidine 0.5mM, triton X-100 0,1%, glycerol 20%, PIC 1X), mix well and incubate on balancelle 10'.   Pellet isolated nuclei at 1400rcf 15' 4°C, wash once in 250mL PBS, 1400g, 10' 4°C Tagmentation: Resuspend isolated nuclei in 50µL tagmentation buffer (33mM tris-acetate pH 7.8, 66mM potassium acetate, 10mM Mg-acetate, 16% dimethylformamide, 0.01% digitonin) + 0.5µL pre-loaded Tn5, mix well. Incubate 60' at 37°C 300rpm.  DNA isolation add 50µL STOP buffer (EDTA 20mM, tris-HCl pH 7.5 10mM) add 200µL H20, mix well,  add 300µL basic Phenol-chloroforme-isoamyl alcool, vortex 10s,  incubate RT 10', centrifuge 15' 160000g 4°C Collect supernatent, add 1V chloroforme, vortex 10s, centrifuge 15' 4°C 160000g Collect supernatent, add 2V Et-OH 100%, 1/10V NaAc 3M, incubate at -20°C ON Centrifuge 16000g 15' 4°, remove supernatent, add 500µL 75% Et-OH, centrifuge 15' 4°C 16000g, repeat twice Air-dry 10', 65°C 5', resuspend in 25µL H20. Amplify purified library :  1. 72°C 5' 2. 98°C 30\" 3. 98°C 10\" 4. 63°C 30\" 5. 72°C 1' 6. Hold 4°C  repeat Step 3-4 14 times