GSM4551899: Adult_Mecp2KO_CTX_IgG_CNR_rep1; Mus musculus; OTHER
Mouse cortices were collected and  homogenized with a dounce-homogenizer and tight pestle 15 times in 5 mL Buffer HB (0.25M sucrose, 25mM KCl, 5mM MgCl2, 20mM Tricine-KOH pH7.8, 1mM DTT, 0.15 mM spermine, 0.5 mM spermidine, Roche protease inhibitor tablets, 10mM Sodium Butyrate, 1mM RNase Inhibitor when doing downstream RNA-seq). IGEPAL CA-630 (Sigma) was added to a final concentration of 0.3% to the homogenate, followed by douncing 5 more times with tight pestle. Homogenized cortices were filtered through a 40um strainer then combined with Working Solution (5 volume of OptiPrep + 1 volume of Diluent [150 mM KCl., 30 mM MgCl2, 120 mM Tricine-KOH pH 7.8] ) at a 1:1 ratio. The mixture was underlaid with 1.2 mL 30% and 1.2 mL 40% OptiPrep gradient solutions (Working solution and diluted with HB Buffer to 30% or 40%). Following ultracentrifugation, 1 mL of nuclei were collected from the interface of the 30% and 40% Optiprep layers. Draq 5 (Abcam) was used to stain DNA. Using a Sony SH800Z Cell sorter, GFP positive nuclei (using Sun1-GFP on nuclear membrane) were selected and sorted into CUT&RUN wash buffer (1 mL of 1M HEPES pH 7.5, 1.5 mL of 5M NaCl, 1 mL of 10% Tween-20, 0.05g BSA, 25 µl of 1M spermidine, protease inhibitors (Roche), H2O to 50 mL). Nuclei were bound to concanavalin A-coated (ConA) beads then washed once with CUT&RUN binding buffer (400 µl of 1M HEPES pH 7.9, 200 µl of 1M KCl, 20 µl of 1M CaCl2, 20 µl of 1M MnCl2, H2O to 50 mL). ConA-bead-bound nuclei were incubated overnight in cold CUT&RUN antibody buffer (1 mL CUT&RUN Triton-wash buffer, 4 µl of 0.5M EDTA) with either in-house anti-FOS antibodies (Emi Ling and Greenberg M.E., in preparation), anti-ARID1A (Cell signaling #12354) or IgG (Cell Signaling #2729S). After incubation, the ConA-bead-bound nuclei were washed once with CUT&RUN antibody buffer, and protein-A-MNase (Skene and Henikoff, 2017) was added to each sample to a final concentration of 700 ng/mL, followed by an incubation for one hour at 4°C. The ConA-bead-bound nuclei were washed twice with CUT&RUN Triton-wash buffer (1 mL of CUT&RUN wash buffer, 10 µl of 10% Triton X-100), and resuspended in 100 µl Triton wash buffer. To each sample, 3 µl of 0.1M CaCl2 was added to each sample, and incubated on ice for 30 min. The reaction was stopped by adding 100 µl 2x STOP buffer (68 µl of 5M NaCl, 40 µl of 0.5M EDTA, 8 µl of EGTA, 4 µl 10% Triton X-100, 2 µl of 1 pg/ul spike-in DNA, 10 µl of RNase A, H2O 868 µl) and incubating at 37°C for 20 minutes. Two µl of 10% SDS was added to the supernatant and the samples were proteinase K treated for > 2 hours at 65°C. DNA was collected by ethanol precipitation. Library preparation was performed as previously described (Hainer and Fazzio, 2019), except that Rapid Ligase (Enzymatics) was used for adapter ligation, and AMPure XP beads were used for the cleanup of the final libraries. All CUT&RUN libraries were paired-end sequenced (40 bases x 2) on a Nextseq 500.
GEO Accession
GSM4551899