title: Illumina HiSeq 2500 sequencing; RNA-Seq of A375 cells treated with tetracycline-based crosslinking probes, with and without competition by parent tetracyclines, to show specific binding of the tetracycline analogs Col-3 and doxycycline on rRNA substructures of the human ribosome
design: RNA-Seq of A375 cells treated with tetracycline-based crosslinking probes, with and without competition by parent tetracyclines, to show specific binding of the tetracycline analogs Col-3 and doxycycline on rRNA substructures of the human ribosome
library_construction_protocol: 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: Experimental Factor: chemical probe
attributes.value: COL-3 probe
attributes.tag: Experimental Factor: compound
attributes.value: none
attributes.tag: Experimental Factor: irradiate
attributes.value: UV radiation 365 nm for 20 min