MassIVE MSV000093210

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MECP2 directly interacts with RNA polymerase II to modulate transcription in human neurons

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Description

This dataset comprises 15 raw AP-MS files, each with an associated peak list, captured using a Vanquish Neo nanoLC system in tandem with an Orbitrap Eclipse mass spectrometer. To generate MECP2 hESC-reporter lines for wild type (WT) and various mutations of MECP2, including R133C, R168X, and R270X, we first used CRISPR/Cas9 to create MECP2 alleles carrying the green fluorescent protein (GFP) sequences in the endogenous gene. The R133C mutation was then introduced into the WT MECP2-GFP reporter line. Mutations R133C, R168X, and R270X are recognized as loss-of-function variants in MECP2 and are also identified as primary Rett syndrome-causing mutations. For efficient neuronal differentiation, a doxycycline (DOX)-responsive NGN2 construct was incorporated at their AAVS1 safe harbor locus. Upon the addition of DOX, homogenous populations of neurons were generated within three weeks from those four MECP2 hESC-reporter lines. Subsequently, GFP-pull down assay and AP-MS were performed using these WT MECP2-GFP neurons along with R133C-, R168X-, and R270X-mutant MECP2-GFP reporter neurons. Neurons expressing only the GFP tag served as a negative control. AP-MS analysis identified proteins interacting differently between WT and mutant MECP2 within human neurons. [doi:10.25345/C5G737F23] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: MECP2 ; DIA

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Principal Investigators:
(in alphabetical order) 		Fabian Schulte, Whitehead Institute, United States
Submitting User: fschulte
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Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

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