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Calcineurin signaling regulates the formation of activity-induced DNA breaks by dephosphorylating Topoisomerase IIB at the nuclear periphery

ABSTRACT: 994820 Fear conditioning: Top2B immunoprecipitated from mouse hippocampus dissected 30 minutes after Fear Conditioning 994821 Control: Top2B immunoprecipitated from hippocampus dissected from mouse that were in home cages. Complete description: Hippocampus brain tissue (4 mouse per sample) from were lysed in in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.2% N-Lauroylsarcosine, phosphatase and protease inhibitors cocktail) by passing through a 25-gauge needle and incubated with Benzonase Nuclease (>=250 units/microliters) 2microliters for 0.3ml of sample for 3hrs followed by sonication. Samples were centrifuged at 20000 x g for 5 min. The supernatants were collected, and protein concentrations determined using the BCA assay. Equal amounts of protein and 6 microliters of Top2B antibody (Rabbit, Abcam, ab125297) per sample were incubated overnight. Samples were incubated with 25 microliters of protein A/G-magnetic beads for 3 h and the beads were washed 4 times with high salt buffer (500 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 0.1% SDS) and one more time with TBS with 0.1% Triton X-100. All procedures were performed at 4C. Beads were re-suspended in Laemmli buffer to separate proteins by electrophoresis.The gel was stained with Coomassie blue and the Topoisomerase 2 band was cut.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Ram Madabhushi

PROVIDER: MSV000089825 | MassIVE | Wed Jul 06 08:49:00 BST 2022

REPOSITORIES: MassIVE

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Project description:Glioblastoma stem cells (TS-543) were harvested and cross-linked with 1% formaldehyde for 10 mins at room temperature. The cells were lysed using SDS Lysis buffer for ChIP (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8). The lysate was then sonicated for 25 cycles at 30% amplitude (15 s ON and 45 s OFF). The sonicated samples were then diluted in ChIP dilution buffer (0.01% SDS, 1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8, 167 mM NaCl) and used for the immunoprecipitation with H3K27ac (Abcam, Ab4729), H2AZ (Active motive, cat#39113) and H3K27ac (Abcam cat#ab8580). After an over-night incubation with antibody, the bound DNA was washed sequentially with low salt wash buffer (0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% NP40, 1%deoxycholate, 1 mM EDTA, 10 mM Tris–HCl pH 8) and TE wash buffer (10 mM Tris–HCl pH 8, 1 mM EDTA) to remove non-specific sequences and eluted in the elution buffer (84 mg NaHCO3, 1 ml 10% SDS, 9 ml H2O). Then the samples were reverse cross-linked using NaCl at 65°C overnight. The eluted DNA was purified and used for library preparation. Library preparation was performed as previously described as described in Bowman et al. BMC Genomics 2013. Briefly, end repair with NEBNext End Repair enzyme (NEB, E6050) and clean-up with 2.4x volume AMPure XP beads (Beckman Coulter, A63880). A-tailing with Klenow Fragment (NEB, M0212) and purified with 2.4x volume AMPure XP beads. Adapter ligation reactions contained annealed universal adapter and T4 rapid ligase (NEB, B0202) and clean-up with 1.8x volume AMPure XP beads. Chromatin was amplified using KAPA Real-time Library Amplification Kit (KAPABIOSYSTEMS, KK2701) with universal primer and barcoded primer. Amplified chromatin was purified with QIAquick Gel Extraction Kit (Qiagen) and sequenced paired-end with Illumina NextSeq 500.

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Calcineurin signaling regulates the formation of activity-induced DNA breaks by dephosphorylating Topoisomerase IIB at the nuclear periphery

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