Detection of various pre-rRNA processing intermediates by combining different linkers and primers. (A) Western blot of TAP purification eluates with different bait proteins as labeled. Antibodies used for the detection of assembly factors or ribosomal proteins are indicated at the right side. (B) Northern blot of RNA isolated from different pre-ribosome pull-down samples. The probes used are indicated on the left, (pre)-rRNA species on the right. (C) Simplified scheme to show at which stages the bait proteins used are involved in ribosome biogenesis. The copurifying pre-rRNA species are also indicated. (Light pink) 90S pre-ribosomal particle/pre-rRNA, (soft blue) 60S pre-ribosomal particle/pre-rRNA, (soft red) 40S pre-ribosomal particle/pre-rRNA. (D–F) 1.5% agarose gel separating the products of the Riboprobing workflow. (m) 100-bp ladder (Thermo Fisher). (D) RNA, isolated from Utp14-TAP and Tsr1-TAP pull-downs, was ligated with the 5′ linker and after cDNA synthesis amplified with 5′ linker and 18S primer. (E) RNA, isolated from Tsr1-TAP and Noc2-TAP pull-downs, was ligated with the 3′ linker and after cDNA synthesis amplified with the 18S primer and 3′ linker. (F) RNA, isolated from Nog1-TAP and Noc2-TAP pull-downs, was ligated with the 3′ linker and after cDNA synthesis amplified with the 5.8S primer and 3′ linker. (G) Riboprobing workflow used in D–F with detectable pre-rRNA species.
