Dataset Information

Immunoprecipitation of Myc-UBL3 from MutuDC1940 cells

ABSTRACT: Ubl3KO MutuDC or Ubl3KO MutuDC stably expressing either Myc-UBL3(WT) or Myc-UBL3(C114S) were lysed in 1% Triton buffer on ice and nuclei removed by centrifugation at 14,000 × g at 4 °C. Postnuclear supernatants of 5 x 10^6 cells were incubated with anti-Myc-Tag (9B11) mouse mAb conjugated to magnetic beads (Cell Signaling Technologies) at 4 ºC, washed and eluted by acidification with 0.1% FA and denatured with 2,2,2-trifluoroethanol. Proteins were reduced with 12.5 mM tris(2-carboxyethyl)phosphine (TCEP), and digested with 0.25 mg proteomics grade Trypsin (Sigma-Aldrich) in 50 mM triethylammonium bicarbonate (TEAB)) overnight at 37 °C. Peptides were dried by SpeedVac, reconstituted in 2% acetonitrile and 0.05% trifluoroacetic acid, centrifuged for 10 minutes at 21,000 × g to remove particulate matter, and filtered over Vivacon 500 30kDa columns (Satrorius).

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Justine Mintern

PROVIDER: PXD030790 | JPOST Repository | Fri Jan 07 00:00:00 GMT 2022

REPOSITORIES: jPOST

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Dataset's files

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			Action	DRS
	210712_UBL3C114S-2603_4.raw	Raw
	210712_UBL3C114S-2603_5.raw	Raw
	210712_UBL3C114S-2603_6.raw	Raw
	210712_UBL3KO-2603_1.raw	Raw
	210712_UBL3KO-2603_2.raw	Raw

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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.

Dataset Information

Immunoprecipitation of Myc-UBL3 from MutuDC1940 cells

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