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PSKH1:UNC119B SDA crosslinking

ABSTRACT: Recombinant proteins were mixed and then crosslinked by SDA and UV exposure. Complexes were resolved by SDS-PAGE, excised and tryptic peptides identified by MS.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Prof. James Murphy

PROVIDER: PXD056476 | JPOST Repository | Thu Jan 30 00:00:00 GMT 2025

REPOSITORIES: jPOST

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Dataset's files

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			Action	DRS
	P5025.fasta	Fasta
	P5025_01.mgf	Mgf
	P5025_01.raw	Raw
	P5025_02.mgf	Mgf
	P5025_02.raw	Raw

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Project description:UV-crosslinking and high througput sequencing of cDNAs (CRAC) was used to map the binding sites Hfq in enterohaemorhaggic E. coli (EHEC). Hfq was tagged with a His-FLAG dual affintiy tag and UV crosslinked after growth in the MEM-HEPES media essentailly as per Granneman et al (2009) PNAS. We additionally crosslinked Hfq in non-pathogenic E. coli K12 str. MG1655 grown in LE media. Hfq-RNA complexes were purified and trimmed using RNase A/T1. RNA fragments were isolated and converted to cDNA, PCR amplified and sequenced using Illumina Solexa GAxII and HiSeq2000 platforms. His-FLAG tagged Hfq and untagged controls were cultured to an OD of 0.8 and crosslinked with UV-C. Five replicates of tagged EHEC Hfq and 2 replicates of untagged Hfq were crosslinked. We have additionally crosslinked 2 replicates each of E. coli K12 tagged and untagged Hfq.

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