GSM6504781: 502F DA735 H3K9me3; Homo sapiens; OTHER
Nuclei extraction: Protocol based on published nuclei isolation protocol (Södersten et al., 2018). Around 20 μg of -80 C-conserved tissue were thawed and dissociated in ice-cold lysis buffer (0.32M sucrose, 5 mM CaCl2, 3 mM MgAc, 0.1 mM Na2EDTA, 10 mM Tris-HCl pH 8.0, 1 mM DTT) using a 1 ml glass douncer (Wheaton). The homogenate was slowly and carefully layered in the centrifuge tubes on top of a sucrose layer (1.8 M sucrose, 3 mM MgAc, 10 mM Tris-HCl pH 8.0, 1 mM DTT) to create a gradient, and then centrifuged at 15500 rpm for 2 h 15 min. Following centrifugation, the supernatant was removed and the pellet softened for 10 minutes in 100 μl of nuclear storage buffer (15% sucrose, 10 mM Tris-HCl pH 7.2, 70 mM KCl, 2 mM MgCl2) prior resuspension in 300 μl of dilution buffer (10 mM Tris-HCl pH 7.2, 70 mM KCl, 2 mM MgCl2, Draq7 1:1000). The suspension was then filtered (70 μm cell strainer) and sorted via FACS (FACS Aria III, BD Biosciences) at 4° C with low flowrate, using a 100 μm nozzle (Pipette tips and Eppendorf tubes for transferring nuclei were pre-coated with 1% BSA). CUT&RUN: We followed the protocol detailed by the Henikoff lab  (Skene et al., 2018)  with some modifications. ConA-coated magnetic beads (Epicypher) were activated by washing twice in bead binding buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl, 1 mM MnCl2) and placed on ice until use. For fetal cortex samples, 300,000 or 600,000 single nuclei were sorted per frozen tissue piece. For adult neuron samples, 300,000 NeuN+ single nuclei were sorted per frozen tissue piece. Pipette tips for transferring nuclei were pre-coated with 1% BSA. Nuclei were pelleted and resuspended in ice-cold nuclear wash buffer (20 mM HEPES, 150 mM NaCl, 0.5 mM spermidine, 1x cOmplete protease inhibitors, 0.1% BSA) and 30 μL (10 μL per antibody treatment) of ConA-coated magnetic beads (Epicypher) added with gentle vortexing. Binding of nuclei to beads proceeded for 10 min at RT with gentle rotation, and then bead-bound nuclei were split into three equal volumes (corresponding to IgG control, H3K4me3 and H3K9me3 treatments). After removal of the wash buffer, nuclei were then resuspended in 100 μL 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 (rabbit anti-H3K9me3, Abcam ab8898, RRID:AB_306848; rabbit anti-H3K4me3 Active Motif 39159, RRID:AB_2615077; or goat  anti-rabbit   IgG,  Abcam  ab97047,  RRID:AB_10681025)  at  1:50  dilution  and incubated at 4 ºC overnight with gentle shaking. Nuclei were washed 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, pA-MNase (a generous gift from Steve Henikoff) was added in nuclear digitonin wash buffer and incubated with the nuclei at 4 ºC for 1 h. Nuclei were washed twice, resuspended in 100 μL digitonin buffer, and chilled to 0-2 ºC in a metal block sitting in wet ice. Genome cleavage was stimulated by addition of 2 mM CaCl2 at 0 ºC for 30 min. The reaction was quenched by addition of 100 μL 2x stop buffer (0.35 M NaCl, 20 mM EDTA, 4mM EGTA, 0.02% digitonin, 50 ng/μL glycogen, 50 ng/μL RNase A, 10 fg/μL yeast spike-in DNA (a generous gift from Steve Henikoff)) and vortexing. After 30 min incubation at 37 ºC to release genomic fragments, bead-bound nuclei were placed on the magnet stand and fragments from the supernatant purified by a NucleoSpin clean-up kit (Macherey-Bagel). Illumina sequencing libraries were prepared using the Hyperprep kit (KAPA) with unique dual-indexed   adapters   (KAPA),   pooled   and   sequenced   on   a Nextseq500   instrument (Illumina).