RNA proximity labeling in log phase Caulobacter crescentus NA1000

APEX2 RNA proximity labeling is a powerful method for determining localized RNAs in vivo. APEX2 RNA proximity labeling was adapted to bacterial cells, using an APEX2 fusion to the core scaffold of BR-bodies, RNase E. As a control, APEX2 was also fused to a variant of RNase E that lacks its C-terminal IDR and is unable to form BR-bodies, RNase E delta CTD. RNA proximity labeling was performed, and we observed a similar pattern of enriched RNAs to density centrifugation isolated BR-bodies (Al-Husini et al. Mol Cell 2020). Duplicate cultures of RNase E-APEX2 and RNase E(delta CTD)-APEX2 strains were growth to mid-log phase, and incubated with alkyne phenol for 30 minutes. The cells were incubated with H2O2 for 1 minute, and then harvested for RNA extraction. An aliquot of the lysate of each biological replicate was saved for normalization, and the proximity labled RNA was isolated by attaching a biotin-azide via click chemistry followed by purificaiton on streptavidin resin. Lysate RNA and proximity labeled RNA samples were then prepared for RNA-seq by the IU CGM core facility.