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PDAP1 RIP in UV-crosslinked human T cells

ABSTRACT: PDAP1 RIP in UV-crosslinked human T cells

PROVIDER: PRJNA761680 | ENA |

REPOSITORIES: ENA

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PDAP1 RIP in UV-crosslinked human T cells

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UPA-Seq: Prediction of functional lncRNAs using the sensitivities to UV-crosslinking

Project description:Assuming that functional lncRNAs form larger ribonucleoprotein complex and thus are easily crosslinked to proteins upon UV-irradiation, we performed RNA-Seq analyses of RNAs recovered from the aqueous phase after the UV-irradiation and phenol chloroform extraction (UPA-Seq)

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UV-crosslinking and high throughput sequencing of cDNAs (CRAC) of the RNA chaperone Hfq in enterohaemorhaggic E. coli

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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