CUT&RUN against H3K4me3 and H3K27me3 in E16 and L3 neurons
CUT&RUN against H3K4me3 and H3K27me3 in E16 and L3 neurons
Embryonic cells were isolated as follows. Staged egg laying was carried out to produce E16 embryos. elav-nRFP and Oregon-R strains were amplified, staged lays were done on yeast apple juice agar at 25°C. After a pre-lay period of 30 min, the agar plates and yeast were replaced with fresh plates and flies were left to lay for 3 hours at 25°C. Agar plates were removed and kept at 25°C, embryos were collected 11 hours and 40 min AEL, when they had reached stage 16. Embryos were then isolated from the medium and washed on a 100 µm mesh. The collected embryos were transferred into a cold solution of phosphate-buffered saline (PBS) in a Dounce homogenizer on ice. The embryos were dissociated using the large clearance pestle then the small clearance pestle, then filtered (70 µm filter).   L3 neurons were purified from elav-nRFP larvae. Larval brains were dissected in cold PBS on ice and transferred to tubes containing 0.5μg of collagenase IV (Gibco, Invitrogen) in 220µL of PBS. Brains were incubated at 37°C on a Thermomixer with shaking at 500 rpm for 20 min. Then the brains were dissociated by pipetting up and down with 10-gauge needles and syringes and filtered through a 70 µm filter.  Cells were sorted using FACS Aria II (BD Biosciences) at 4°C in three independent biological replicates for each genotype. Live cells were first selected based on the forward scatter and side scatter and only single cells were sorted according to the RFP signal. Oregon-R cells were used as a negative control to set the gate and sort the RFP positive cells  After sorting cells were washed in PBS (600 g 5 min, 4°C) and nuclei were extracted in triton buffer (20 mM HEPES-KOH pH 7.9, KCl 10 mM, spermidine 0.5 mM, triton-X-100 0.1%, glycerol 20%, protease inhibitor (Sigma Aldrich #5056489001)) for 10 min at 4°C on a rotating platform. Extracted nuclei were washed once in 1V PBS (1300 g, 5 min, 4°C) and fixed in paraformaldehyde (PFA) (1% final) 2 min at RT. PFA was quenched by the addition of 2.5 M glycine (5 min at RT). Nuclei were washed 3 times in 1 mL binding buffer (HEPES-KOH pH 7.9 20 mM, KCl 10 mM, CaCl2 1 mM, MnCl2 1 mM, protease inhibitor) and incubated for 10 min on a rotating platform at 4°C with 1 mL binding buffer containing 10µl magnetic concanavalin A-coated beads (Bangs Laboratories #BP531) per sample. Bead-bound nuclei were then handled on a magnet stand.  Nuclei were washed once in EDTA buffer (wash buffer supplemented with 0.2 mM EDTA) and 3 times in wash buffer (HEPES-KOH pH 79 20 mM, NaCl 150 mM, Spermidine tetrahydrochloride 0.5 mM, BSA 0.1%, protease inhibitor). Nuclei were resuspended in 50µl wash buffer per sample, split in 6 samples, supplemented with 1µl primary antibody (H3K27me3: cell signaling 9733, H3K4me3: abcam ab8580, rabbit anti-mouse IgG abcam ab46540) and incubated overnight on a rotating platform at 4°C. The next day, nuclei were washed 3 times in wash buffer, incubated 1h at RT with a secondary antibody (Guinea-pig anti-Rabbit IgG ABIN101961), washed again 3 times in wash buffer, resuspend in 100µl wash buffer supplemented with pA-MNase at 0.7 ng/mL final concentration) and incubated 1h at 4°C. Nuclei were than washed 3 times in wash buffer, resuspend in 300µl wash buffer containing 1mM CaCl2 to induce the digestion and incubated 30 min on ice cold water. Reaction was stop by adding 150µl EDTA buffer for 20 min at 37°C. Crosslink and MNase were digested for 10 min at 70°C by addition of SDS (0.05% final concentration) and Proteinase K (0.6 mg/mL final concentration). DNA was extracted using phenol/chloroform/isoamyl alcohol extraction. Precipitation of DNA was performed with addition of glycogen (3 µg/mL final concentration).  Library preparation was performed using NEBNext®Ultra II DNA Library Prep Kit for Illumina® using following protocol with 14 amplification cycles: (dx.doi.org/10.17504/protocols.io.wvgfe3w).