title: Biomolecular Condensate Coronas from Nanoparticle Intracellular Processing Are Transferred Between Cells Allowing Cytosolic and Nuclear Access
design: Biomolecular Condensate Coronas from Nanoparticle Intracellular Processing Are Transferred Between Cells Allowing Cytosolic and Nuclear Access
library_construction_protocol: HEK293 cells were seeded into T175 flask and cultured for 24 h. The culture medium was then removed, the cells were washed twice with pre-warmed PBS, and pre-warmed serum-free medium was added before incubating for a further 24 h. The conditioned serum-free medium was collected and centrifuged at 2,000 × g for 30 mins at 4 ℃ to remove cells, followed by 16,000 × g for 20 mins at 4 ℃ to remove cell debris. The resulting supernatant was ultracentrifuged at 160,000 × g for 1.5 h at 4 ℃. The pellet was resuspended in PBS and ultracentrifuged again at 160,000 × g for 2 h at 4 ℃. The final pellet was used for proteomics analysis.  The supernatant were ultracentrifuged at 160,000 g for 1.5h HEK293 cells are cultured in complete growth media Complete growth media were removed. HEK293 cells were treated with serum-free media for 24h 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.