Dataset Information

USP8 maintains ESC stemness via deubiquitination of EPG5

ABSTRACT: The coiled-coil domain of EPG5 was synthesized as follows: Biotin-MNKNEAVSQQIHVLQKEVRQLQAE AAQ (from Beijing SciLight Biotechnology, LCC). 1 mg of this short peptide was incubated with ES cells lysates followed by immunoprecipitation with anti-biotin beads. The precipitates were extensively washed with lysis buffer then boiled with SDS loading buffer and subjected to SDS–PAGE. Total proteins were excised from the gels, then subjected to in-gel digestion and analysis by LC-MS/MS.

INSTRUMENT(S): LTQ Orbitrap XL

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture, Embryonic Stem Cell

DISEASE(S): Disease Free

SUBMITTER: xingxing shi

LAB HEAD: i.e.Tongbiao Zhao

PROVIDER: PXD012563 | Pride | 2019-02-25

REPOSITORIES: Pride

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USP8 maintains embryonic stem cell stemness via deubiquitination of EPG5.

Gu Haifeng H Shi Xingxing X Liu Chao C Wang Chaoqun C Sui Ning N Zhao Yu Y Gong Jiaqi J Wang Fuping F Zhang Hong H Li Wei W Zhao Tongbiao T

Nature communications 20190401 1

Embryonic stem cells (ESCs) can propagate in an undifferentiated state indefinitely in culture and retain the potential to differentiate into any somatic lineage as well as germ cells. The catabolic process autophagy has been reported to be involved in ESC identity regulation, but the underlying mechanism is still largely unknown. Here we show that EPG5, a eukaryotic-specific autophagy regulator which mediates autophagosome/lysosome fusion, is highly expressed in ESCs and contributes to ESC iden ...[more]

PMID: 30931944

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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.

			Action	DRS
	BPI_7601_DUIZHAO.raw	Raw
	BPI_7601_DUIZHAO_____03654.xlsx	Xlsx
	BPI_7601_YANGPING.raw	Raw
	BPI_7601_YANGPING.xls	Xls
	F003654_DUIZHAO.dat	Other