GSM5693323: dat_8mo_D834_wt_K27; Mus musculus; ChIP-Seq
Midbrain dopamine producing neurons (mDA) from DATcre-Eed-Rpl10a-mCherry mice were extracted. Tissue was thawed and dissociated using a 1 mL dounce homogenizer (Wheaton) in ice-cold lysis buffer (0.32M sucrose, 5mM CaCl2, 3 mM MgAc, 0.1 mM Na2EDTA, 10 mM Tris-HCl, pH 8.0, 1 mM DTT, 1x complete proteinase inhibitor, EDTA-free (Roche)). The homogenate was layered over a sucrose cushion (1.8M sucrose, 3mM MgAc, 10 mM Tris-HCl, pH 8.0, 1 mM DTT) before centrifugation for 2.2h at 30.000g in a Beckman J-25 centrifuge using a J13.1 rotor. The pelleted nuclei were re-suspended in a nuclear storage buffer (15% sucrose, 10 mM Tris_HCl, pH 7.2, 70 mM KCl, 2 mM MgCl2) supplemented with RNAse inhibitor (RNAse out, Invitrogen) and proteinase inhibitor (Complete, Roche). Re-suspended nuclei were passed through a 30 μm cup filcon (BD Biosciences, 340625) into a BSA-coated tube for sorting. FACS was performed using a FACSAria cell sorter and the FACSDiva software (BD Biosciences). The nuclei were identified by forward and side scatter gating and a 633 nm laser with a 660/20 or 616/23 filter. A 100 μm nozzle, sheath pressure of 20–25 psi, and an acquisition rate of up to 1000 events per second were used. Cell-type specific nuclei were collected in batches of 1000, supplemented with a nuclear buffer (10 mM Tris (pH 8.0), 100 mM NaCl, 2 mM MgCl2, 0.3M Sucrose, 0.25% IGEPAL CA-630) to a total volume of 10 uL and subsequently snap frozen on dry ice for later use. ChIP and library generation for ChIP-seq was performed using the previously described ULI-NChIP protocol [20] with minor modifications. Briefly, one batch of 1000 nuclei per animal and antibody was thawed on ice and fragmented for 10 min using MNase at 21 °C and diluted in NChIP immunoprecipitation buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 15 mM NaCl, 0.1% Triton X-100, 1x EDTA-free protease inhibitor cocktail and 1 mM phenylmethanesulfonyl fluoride (PMSF, Sigma, P7626)). Chromatin was pre-cleared with 5 μl of a 1:1 mixture of Protein A and Protein G Dynabeads (Life Technologies, 10006D and 10007D) and IPed over night at 4 °C with 0.25 of anti-H3K9me3 (Active Motif, 39161), anti-H3K27me3 (Cell Signaling, 9733) or anti-H3K4me3 (Cell Signaling, 9751). Beads were washed twice with 400 μl of a buffer containing 20 mM Tris-HCl, pH 8.0, 0.1% SDS, 1% Triton X-100, 0.1% deoxycholate, 2 mM EDTA and 150 mM NaCl and twice with a buffer containing 20 mM Tris-HCl (pH 8.0), 0.1% SDS, 1% Triton X-100, 0.1% deoxycholate, 2 mM EDTA and 500 mM NaCl. Protein–DNA complexes were eluted in 30 μl of a ChIP elution buffer containing 100 mM NaHCO3 and 1% SDS for 2 h at 68 °C. The eluted chromatin was purified by phenol chloroform, precipitated with EtOH and re-suspended in 10 mM Tris-HCl pH 8.0. To generate libraries for sequencing, ChIPed DNA was end-repaired in a mixture of dNTP (NEB, N0447L), T4 DNA polymerase (NEB, M0203L), Klenow DNA polymerase (NEB, M0210L) and T4 PNK (NEB, M0201L) in T4 DNA ligase buffer (NEB, B0202S) for 30 min at room temperature. The product was purified with 1.8x Agencourt AMPure XP beads (Beckman Coulter, A63881) and then poly-A tailed in dATP (NEB, N0440S) and Klenow (3'-5' exo-) (NEB, M0212L) in NEB buffer 2 (NEB, B7002S) at 37 °C for 30 min followed by another 1.8x AMPure bead purification step. The product was ligated to NEBnext adaptors for Illumina (NEB, E7337A) with Quick DNA ligase (NEB, M2200L) in 2x Quick DNA ligation buffer at room temperature for 1-2h, trimmed with USER enzyme (NEB, E7338A) at 37 °C for 15 min and subsequently purified with 0.8x AMPure beads. The adapter-ligated ChIP-DNA was then PCR-amplified using 2x Phusion HF Master mix (NEB, M0531L) and NEBNext Multiplex Oligos for Illumina, Index Primers Set 1 (NEB, E7335L). The final libraries were cleaned up using 0.8x AMPure beads, quantified on a Qubit 3.0 Fluorometer (ThermoFisher) and quality checked on a 2100 Bioanalyzer system (Agilent) using High Sensitivity DNA kit (Agilent, 5067-4626).