Fig. 1

Isolation of RNA crosslinked to the exosome of S. solfataricus. a Autoradiogram of a nitrocellulose membrane with transferred, coimmunoprecipitated proteins, attached to crosslinked, radioactively labeled RNA. Archaeal cells were subjected to UV irradiation (+ UV) or not (− UV) to crosslink RNA to RNA binding proteins in vivo. After lysis, the cleared lysate was used for CoIP with antibodies directed against the proteins indicated above. After extensive washing with buffer containing 1 M NaCl, the coimmunoprecipitated, crosslinked RNA was radioactively labeled directly on the beads. The coimmunoprecipitated proteins with crosslinked RNA were separated by SDS-PAGE and transferred to the membrane. The uncropped image is shown in Fig. S1 (Additional file 1). b Western blot analysis of the nitrocellulose membrane shown in a) with antibodies directed against aRrp41. On the right side of the gel, recombinant, His₆-tagged aRrp41 was loaded as positive control (cropped in panel a; see Fig. S1). Here, a non-blotted gel lane with marker proteins (run in the same gel) is shown on the left. c Representative growth curve of S. solfataricus in a 10 l bioreactor. Cells were harvested for iCLIP at OD₆₀₀ of 0.7 (marked with an arrow). d Autoradiogram of the nitrocellulose membrane with samples used in our iCLIP analysis. Harvested cells were UV-irradiated, divided into 3 portions and subjected to CoIP with antibodies specific to aRrp41, aRrp4 and Trx. Two biological replicates were performed. After CoIP, 3′-RNA linker ligation and radioactive labeling of bound RNA, protein-RNA complexes were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The autoradiogram was used to determine the membrane areas of the protein/RNA complexes (marked on the right side), which were excised and used for iCLIP library preparation and sequencing. The uncropped image is shown in Fig. S1 (Additional file 1)