organism: Homo sapiens
description: Protocols: We incorporated dual bioorthogonal handles into tetracycline-based probes, containing both a photoactive diazirine to enable direct probe crosslinking to the human ribosome and an azide handle to allow selective enrichment of crosslinked biomolecules via copper-free 'click' chemistry. Following compound treatment (the probes were used at 20uM concentration and their drug competitors, COL-3 or doxycycline, at 100uM concentration; DMSO was used in control samples) and UV irradiation - 365 nm/20 min (to induce photolysis of the diazirine moiety and subsequent crosslinking to adjacent ribosomal components), cells were directly lysed in dishes with Ribozol reagent. Direct-zol RNA purification kit (Zymo Research) Total RNA was subjected to fragmentation, conjugation of desthiobiotin using 'click' chemistry, ligation of a 3'-adapter, reverse transcription to cDNA, capture and enrichment with streptavidin beads, and PCR amplification.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2018-08-31T17:03:40Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-08-21T12:01:53Z
attributes.tag: External Id
attributes.value: SAMEA4846852
attributes.tag: INSDC center name
attributes.value: HARVARD UNIVERSITY
attributes.tag: INSDC first public
attributes.value: 2018-08-31T17:03:40Z
attributes.tag: INSDC last update
attributes.value: 2018-08-21T12:01:53Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7161:Sample 8
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: A375
attributes.tag: cell type
attributes.value: epithelial cell
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: malignant melanoma
attributes.tag: organism part
attributes.value: skin
attributes.tag: sample name
attributes.value: E-MTAB-7161:Sample 8
attributes.tag: scientific_name
attributes.value: Homo sapiens