alias: GSM4811929
title: GSM4811929: CUTRUN_CA1_CaMK2a_KA2_IgG; Mus musculus; OTHER
library_construction_protocol: Hippocampal tissue from mice was rapidly dissected and dounce homogenized. Dounce homogenization was performed in Buffer HB (0.25M sucrose, 25mM KCl, 5mM MgCl2, 20mM Tricine-KOH, pH 7.8 supplemented with protease inhibitors, 1mM DTT, 0.15mM spermine and 0.5mM spermidine) with tight pestle for 20 strokes in a total of 1.5mL volume. Tissue was then supplemented with 96uL 5% IGEPAL CA-630 and dounced an additional 5 strokes with tight pestle. Homogenate was then filtered through a 40-micron strainer to remove large debris and collected in a 15mL conical. 3.5mL of Buffer HB and 5mL of working solution (50% iodixanol with 25mM KCl, 5mM MgCl2, 20mM Tricine-KOH pH 7.8 supplemented with protease inhibitors, DTT, spermine and spermidine) was added. Homogenized tissue was underlaid with 1mL of 30% iodixanol and 1mL of 40% iodixanol (diluted from working solution) solutions. Samples were centrifuged at 10,000xg for 18 minutes. Nuclei were collected in a 250uL volume at the 30/40% iodixanol interface. Isolated nuclei were diluted two-fold with CUT&RUN wash buffer supplemented with 4mM EDTA and stained with DRAQ5 (Abcam ab108410) at a 1:500 dilution. CaMK2a+ (GFP+) nuclei, resulting from CaMK2a-Cre-mediated expression of Sun1-sfGFP-Myc, were isolated using flow cytometry using a Sony SH800Z Cell Sorter. Sorted nuclei were resuspended in 1 mL cold CUT&RUN wash buffer (20mM HEPES pH 7.5, 150mM NaCl, 0.2% Tween-20, 1mg/mL BSA, 10mM sodium butyrate, and 0.5mM spermidine supplemented with protease inhibitors), using 50,000 nuclei for each reaction. Nuclei were bound to magnetic Concanavalin-A (ConA) beads (Bangs Laboratories) that had been washed with CUT&RUN binding buffer (20mM HEPES-KOH pH 7.9, 10mM KCl, 1mM CaCl2, 1mM MnCl2). ConA-bead-bound nuclei were then incubated overnight in cold CUT&RUN antibody buffer (CUT&RUN wash buffer supplemented with 0.1% Triton X-100 and 2mM EDTA) and an in-house polyclonal Fos antibody (affinity eluted #1096) or rabbit IgG antibody (Cell Signaling Technologies # 2729). After antibody incubation, ConA-bead-bound nuclei were washed with CUT&RUN antibody buffer, resuspended in CUT&RUN Triton-wash buffer (CUT&RUN wash buffer supplemented with 0.1% Triton X-100), and Protein-A-MNase was added at a final concentration of 700ng/mL. Samples were incubated at 4°C for 1h. The ConA-bead-bound nuclei were then washed twice with CUT&RUN Triton-wash buffer and ultimately resuspended in 100uL of CUT&RUN Triton-wash buffer. 3uL of 100mM CaCl2 was added to each sample and samples were incubated on ice for 30 min. The reaction was stopped by adding 100uL of 2x STOP buffer (340mM NaCl, 20mM EDTA, 4mM EGTA, 0.04% Triton X-100, 20pg/mL yeast spike-in DNA, and 0.1ug/mL RNase A) and incubation at 37°C for 20 min. After incubation, ConA beads were captured using a magnet and supernatants containing DNA fragments released by Protein-A-MNase were collected. Supernatants were then treated with 2uL of 10% SDS and 2uL of 20mg/mL Proteinase-K and incubated at 65°C with gentle shaking for 1h. DNA was then purified using standard phenol/chloroform extraction with ethanol precipitation. DNA was resuspended in 30uL of 0.1x TE buffer. CUT&RUN sequencing libraries were generated essentially as previously described (Hainer and Fazzio, Curr. Protoc. Mol. Biol., 2019), with the following modifications: Adapter ligation to end-repaired, A-tailed DNA was performed using Rapid T4 DNA ligase (Enzymatics). PCR-amplified libraries were purified from adapter dimers using a 1.1x ratio of AMPure XP beads, eluting in 20uL of 10mM Tris pH 8.0.
identifiers.namespace: GEO
identifiers.id: GSM4811929
attributes.tag: GEO Accession
attributes.value: GSM4811929