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Characterization of chromatin associated RNA-binding proteins by SPACE

ABSTRACT: Chromatin processes such as DNA replication, transcription and RNA processing are mediated by intermolecular transactions among proteins, RNAs and the genome. Although DNA-protein and RNA-protein interactions have been studied extensively, reliable and efficient identification of chromatin-associated RNA-binding proteins (caRBPs) has remained technically challenging; especially using limited sample material. Here, we present SPACE (Silica Particle Assisted Chromatin Enrichment), a stringent and straightforward chromatin-purification method, which works efficiently with as few as 100,000 cells. Using SPACE we have enriched 1825 chromatin-associated proteins in mES cells. Among many known and novel chromatin-associated proteins, we identified 743 RBPs. Considering protein counts RBPs consist 40% of the chromatin composition, while, weighted by their abundances they comprise ~70% of the chromatome. Furthermore, using a double tryptic digestion strategy (SPACE2) we have determined how the caRBPs bind to chromatin. By applying SPACE to mES cells in 2i and serum conditions, we have detected significant changes in the chromatome of the cells that are neglected in traditional full proteome analyses. Overall, SPACE and SPACE2 are sensitive and powerful methods for providing a global view of chromatin composition and RBP-chromatin interactions.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Embryonic Stem Cell

SUBMITTER: Mahmoudreza Rafiee

LAB HEAD: Nicholas Luscombe

PROVIDER: PXD023903 | Pride | 2021-11-25

REPOSITORIES: Pride

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			Action	DRS
	BCB9922_SPACE_R1_CLF.raw	Raw
	BCB9922_SPACE_R1_RF.raw	Raw
	BCB9922_SPACE_R2_CLF.raw	Raw
	BCB9922_SPACE_R2_RF.raw	Raw
	BCB9922_SPACE_R3_CLF.raw	Raw

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