Biomolecular Condensate Coronas from Nanoparticle Intracellular Processing Are Transferred Between Cells Allowing Cytosolic and Nuclear Access
Biomolecular Condensate Coronas from Nanoparticle Intracellular Processing Are Transferred Between Cells Allowing Cytosolic and Nuclear Access
Cells were exposed to nanoparticle suspension for 10 min (pulse). The suspension was removed. Cells then were incubated complete medium containing FBS for 4 h (chase). After the chase, the supernatants were collected and clarified to remove cell debris. The exported particle complexes were pelleted by centrifugation at 12,000 x g for 40 min at 4 °C.  HEK293 cells are cultured in complete growth media The cells were treated with nanoparticles for 10 min pulse followed by 4 hour chase. The conditioned media were collected. Samples were initially lysed by adding 700 µL QIAzol Lysis Reagent (QIAGEN, Cat. No. 79306), followed by vortexing for 60 sec and incubation at RT (15–25 °C) for 5 min. Subsequently, 140 µL chloroform was added, and samples were shaken vigorously for 15 s. Phase separation was achieved by centrifugation at 14,000 × g for 5 min at RT using QIAGEN MaXtract High Density Tubes. The aqueous phase containing total RNA was collected, and the protocol described in Appendix A of the miRNeasy Micro Kit manual was followed to selectively recover large RNA species (>200 nucleotides) depleted from small RNAs including miRNAs. We used Invitrogen™ Collibri™ Stranded RNA Library Prep Kit for Illumina sequencing.