GoKawiilTissue collection and processing
Human brain tissue samples (GW16–20) were collected from de-identified donors with previous patient consent in strict observance of legal and institutional ethical regulations. All protocols were approved by the Human Gamete, Embryo and Stem Cell Research Committee (GESCRC) and Institutional Review Board (IRB) at the University of California, San Francisco (UCSF). Primary human brain tissue was collected and processed as previously described41. In brief, cortical tissue was collected in artificial cerebrospinal fluid (ACSF) containing 125 mM NaCl, 2.5 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 and 1.25 mM NaH 2 PO 4 under a stereotaxic dissection microscope (Leica). Tissue samples were cut into small pieces and snap-frozen by placing the tissue onto a strip of aluminium foil in prechilled 2-methylbutane on dry ice for around 10 min. The samples were stored in cryovials at −80 °C. For individual germinal zone and cortical plate cultures, the samples were dissected in artificial CSF to separate germinal zone from the cortical plate before dissociation. Tissue pieces were then placed into a prewarmed (37 °C) solution of papain (Worthington) and incubated at 37 °C. After incubation for approximately 60 min, tissue was triturated according to the manufacturer’s protocol, and the samples were spun through an ovomucoid gradient to remove debris. The dissociation medium was removed, and cells were resuspended in NES medium (DMEM/F12 (Gibco), 1:1,000; B27 (Invitrogen), 1:100; N2 (Life Technologies); 20 ng ml−1 FGF (PeproTech); 20 ng ml−1 EGF (PeproTech); 20 µg ml−1 insulin (Thermo Fisher Scientific); 5 ng ml−1 of BDNF (PeproTech); and 10 µM ROCK inhibitor Y-27632 (Selleckchem)). Cells were plated in a Matrigel-coated (1 µg ml−1, Corning) eight-well chambered coverglass (Thermo Fisher Scientific) and allowed to attach for 24 h at 37 °C.
Maintenance, neural induction and neuronal differentiation of iPS cells
The hiPS cell line WTC11 (ref. 42) was obtained from WiCell Stem Cell bank and authenticated by short-tandem-repeat profiling at the source. Cells were maintained in StemFlex medium (Gibco) on plates coated with Matrigel (Corning) and passaged as clumps with ReLeSR (StemCell Technologies). Differentiation of iPS cells was performed as described previously11 with modifications. For neural induction of iPS cells into NPCs, cells were dissociated with Accutase (Gibco) and seeded on Matrigel-coated plates at a density of 150,000 cells per cm2 in StemFlex supplemented with 10 µM Y-27632. The next day (day 0), cells were washed in PBS and fed with neural induction medium (NIM, DMEM/F12 GlutaMax (Gibco) containing 1% ITS-G (Gibco) and 200 µM l-ascorbic acid 2-phosphate (Santa Cruz)). On days 0–2, cells were fed daily with NIM supplemented with 100 nM LDN-193189 (SelleckChem), 10 µM SB431542 (Selleckchem) and 2 µM XAV939 (Cayman). On days 3–9, NIM was supplemented with LDN-193189 and SB431542 only. From day 10 to day 19, cells were fed daily with NPC maturation medium (1:1 mixture of DMEM/F12 GlutaMax and Neurobasal-A (Gibco) supplemented with 1% N-2 and 2% B-27 minus vitamin A (Gibco)). On day 20, NPCs were either collected for analysis or further differentiated into neurons. For neuronal differentiation, NPCs were dissociated with Accutase and seeded at a density of 275,000 cells per cm2 on plates coated with 100 µg ml−1 poly-l-ornithine, 100 µg ml−1 poly-d-lysine, 10 µg ml−1 laminin and 10 µg ml−1 fibronectin (Sigma-Aldrich) in neuronal differentiation medium (Neurobasal-A supplemented with 2% B-27, 2 mM l-glutamine (Gibco), 200 µM l-ascorbic acid 2-phosphate, 10 µM dibutyryl-cyclic AMP (Sigma-Aldrich), 10 ng ml−1 BDNF (Peprotech), 10 ng ml−1 GDNF (Peprotech) and 10 µM DAPT (MedChemExpress)). Cells were fed by 50% medium exchange every 2 days and collected for downstream analysis after 1 week of neuronal differentiation.
EZH2 inhibition in NPCs
On day 10, iPS-cell-derived NPCs were passaged using Accutase and plated onto Matrigel-coated six-well plates in NPC maturation medium supplemented with 10 µM ROCKi. On days 11–12, cells were fed with NPC maturation medium. On day 13, the medium was removed and fresh medium containing EZH2 inhibitor tazemetostat (EPZ-6438, Selleckchem, 1 μM) or DMSO was added to the cells. Cells were fed fresh medium containing the inhibitor (or DMSO) every day for 1 week, at which point they were collected for downstream experimental analyses.
Nucleus isolation and FANS
Snap-frozen cortical tissue was cut into small pieces and transferred to a prechilled 7 ml Dounce Tissue Grinder (Wheaton) containing 5 ml nucleus extraction buffer (NEB: 10 mM HEPES pH 7.4, 25 mM KCl, 5 mM MgCl 2 , 0.25 M sucrose, 0.1% Triton X-100, 1× Halt protease inhibitor cocktail (Thermo Fisher Scientific)). RiboLock (Thermo Fisher Scientific) was added to all the buffers to preserve RNA integrity. While still on ice, the tissue was dissociated with 5–6 strokes with loose pestle A followed by 8–10 strokes with tight pestle B, until no tissue pieces were visible. The sample was incubated on ice for 5 min after which it was transferred to a prechilled 15-ml conical tube. The sample was centrifuged at 500g for 10 min at 4 °C. The supernatant was removed, and the nuclear pellet was resuspended in 10 ml NEB without Triton X-100. The homogenate was passed through a 40 μm strainer into a 50 ml conical tube. Formaldehyde was added to a final concentration of 0.1% and the sample was incubated for 2 min at room temperature with rotation. Glycine was added to a final concentration of 75 mM to quench the reaction. BSA was added to a final concentration of 1% and the sample was centrifuged at 500g for 10 min at 4 °C. The supernatant was discarded, and the nuclear pellet was resuspended in 1 ml staining buffer (PBS containing 1% BSA). The nuclei were filtered through a 40 µm strainer into a 1.5 ml low-bind microcentrifuge tube and counted under a microscope. The nuclei were pelleted at 500g for 10 min at 4 °C and resuspended in 100–150 μl of staining buffer. Antibody staining was carried out for 1 h at 4 °C with rotation using the following antibodies: PAX6-Alexa Fluor 488 (BD Biosciences, 1:20), EOMES-PE-Cy7 (Invitrogen, 1:20) and SATB2-Alexa Fluor 647 (Abcam, 1:100). After staining, 900 μl of staining buffer was added and the samples were centrifuged at 500g for 10 min at 4 °C. The nuclear pellet was resuspended in 1 ml of staining buffer and centrifuged at 500g for 10 min at 4 °C. Nuclei were resuspended in 1–2 ml of staining buffer (depending on the starting material and yield) and filtered into a 70 µm mesh FACS tube (BD). DAPI was added at 1 μg ml−1 just before FANS. AbC Total Compensation capture beads (Thermo Fisher Scientific) were used for generating single-colour compensation controls. FANS was conducted on BD FACS Aria II Cytometer using a 70 μm nozzle. Sorted nuclei were collected in 5 ml tubes containing 300–500 μl of collection buffer (PBS containing 5% BSA and RNasin Plus RNase inhibitor (Promega)). Sorted nuclei were collected by centrifuging at 500g for 10 min at 4 °C and processed for downstream analyses (RNA-seq, Fab blocking and GO-CaRT/CUT&RUN).
Development of Fab blocking for antibody-based chromatin profiling
HEK293T cells were obtained from ATCC and authenticated at source. Cells were collected by trypsinization. Cells were fixed in 0.1% formaldehyde for 2 min at room temperature, followed by quenching in 0.1 M glycine (prepared in wash buffer). Cells were resuspended in 1 ml wash buffer (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.1% BSA, 0.5 mM spermidine and 1× Halt protease inhibitor cocktail) and centrifuged at 300g for 5 min. This step was repeated for a total of two washes. Cells were resuspended in wash buffer and aliquoted at 100 μl per tube into 0.5 ml PCR tubes (around 100,000 cells per experimental condition). Next, 8 μl of BioMagPlus concanavalin A beads (Polysciences) activated in binding buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1 mM CaCl 2 and 1 mM MnCl 2 ) were added to each sample and rotated on a nutator for 10 min at room temperature. The cells were placed onto a magnet to clear and the liquid was removed. Cells bound to beads were resuspended in 50 μl cell permeabilization buffer (20 mM HEPES pH 7.5, 0.1 mM CaCl 2 , 3 mM MgCl 2 , 100 mM KCl, 0.05% digitonin (Sigma-Aldrich) and 1× Halt protease inhibitor cocktail) and incubated for 30 min at room temperature on a nutator. The supernatant was removed, and the beads were resuspended in 50 μl of antibody binding buffer (wash buffer containing 2 mM EDTA and 0.025% digitonin) containing an antibody against H3K27me3 (Cell Signaling Technologies, 1:100). IgG (Cell Signaling Technologies, 1:100) was included in parallel as a negative control. The samples were incubated overnight at 4 °C on a nutator. The next day, the supernatant was removed, and the beads were washed twice with 200 μl wash buffer containing 0.025% digitonin (Wash-Dig). The beads were resuspended in 50 μl of Wash-Dig containing monovalent anti-rabbit Fab fragments (Jackson Immuno) and incubated for 30 min at 4 °C. We tested the following Fab dilutions: 1:20, 1:50, 1:100, 1:250, 1:500, 1:1,000, 1:10,000, 1:50,000 and 1:100,000. We also tested three incubation times: 5 min, 15 min and 30 min. Dilutions of 1:250 and lower and incubation times of 5 min and below did not result in efficient blocking by the Fab fragments. After two washes with WaB-Dig (wash buffer containing 0.025% digitonin), the beads were resuspended in 50 μl of WaB-Dig containing pA/G-MNase (purified from Addgene plasmid 123461 at Macro Lab UC Berkley) and nutated at 4 °C for 1 h. The supernatant was removed, and the beads were washed twice with WaB-Dig. The beads were resuspended in 100 μl of WaB-Dig containing 2 mM CaCl 2 to activate pA/G-MNase. The digestion was carried out for 30 min in a chilled metal block on ice. Digestion was stopped by adding 100 μl of 2× STOP (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 μg ml−1 RNase A, 40 μg ml−1 glycogen and 10 pg ml−1 heterologous DNA). The samples were incubated at 37 °C for 30 min to release pA/G-MNase-cleaved fragments. The contents of the tube were transferred to 1.5 ml Eppendorf tubes containing 2 μl each of 10% SDS and proteinase K (20 mg ml−1). The samples were incubated at 55 °C for 1 h to reverse cross-linking. DNA was extracted using the phenol–chloroform method. Purified DNA fragments were analysed by TapeStation High Sensitivity D1000 assay (Agilent).
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