CUT and RUN

Flash-freeze postmortem brain tissue

Sample 50mg - 100mg from human brain tissue and store at -80°C until use

Activate ConA-coat magnetic beads (Epicypher) by washing twice in bead binding buffer [20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl, 1 mM MnCl₂]. Place on ice until use.

Isolate nuclei from frozen tissue after incubating with Recombinant Alexa Fluor® 488 Anti-NeuN antibody [EPR12763] - Neuronal Marker (ab190195) at a concentration of 1:500 for 30 minutes on ice.

Run nuclei through the FACS at 4°C with low flowrate using a 100 mm nozzle and isolate 300.000 nuclei Alexa Fluor – 488 positive nuclei.

Pellet the sorted nuclei at 1,300 x g for 0h 15m 0s and resuspend in 1mL of ice-cold nuclear wash buffer (20 mM HEPES, 150 mM NaCl, 0.5 mM spermidine, 1x cOmplete protease inhibitors, 0.1% BSA) and 10µL per antibody treatment of ConA-coated magnetic beads (Epicypher) added with gentle vortexing (Pipette tips for transferring nuclei were pre-coated with 1% BSA).

Bind nuclei to beads for 0h 10m 0s at RT with gentle rotation, and then split bead-bound nuclei into three equal volumes (corresponding to IgG control, H3K4me3 and H3K9me3 treatments).

Remove wash buffer and resuspend nuclei in 100µL cold nuclear antibody buffer (20 mM HEPES pH 7.5, 0.15 M NaCl, 0.5 mM Spermidine, 1x Roche complete protease inhibitors, 0.02% w/v digitonin, 0.1% BSA, 2 mM EDTA) containing primary antibody at 1:50 dilution and incubate at 4°C 0h 10m 0s with gentle shaking.

Wash nuclei thoroughly with nuclear digitonin wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x Roche cOmplete protease inhibitors, 0.02% digitonin, 0.1% BSA) on the magnetic stand.

After the final wash, add pA-MNase in nuclear digitonin wash buffer and incubate with the nuclei at 4°C for 1h 0m 0s. Wash nuclei twice, resuspend in 100µL digitonin buffer, and chill to 0°C -2°C in a metal block sitting in wet ice.

Stimulate genome cleavage by addition of 2 mM CaCl₂ at 0 ºC for 30 min. Quench the reaction by additing 100µL 2x stop buffer (0.35 M NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% digitonin, 50 ng/µL glycogen, 50 ng/µL RNase A, 10 fg/µL yeast spike-in DNA) and vortex.

Incubate 0h 30m 0s at 37°C to release genomic fragments. Place bead-bound nuclei on the magnet stand and purify fragments from the supernatant using a NucleoSpin clean-up kit (Macherey-Bagel).

Prepare Illumina sequencing libraries using the Hyperprep kit (KAPA) with unique dual-indexed adapters (KAPA).