title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion in extracellular vesicles
design: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion in extracellular vesicles
library_construction_protocol: BT549 WT cells were seeded as recipient cells. EVs (extracellular vesicles) from scramble control EVs and EFEMP1 kd EVs were used to treat the WT cells for 24 hours. And then the cell pellets were collected. RNA was extracted using the RNA plus isolation kit (Qiagen, 74136) following the protocols. RNA quality control (QC) involved concentration assessment using Nanodrop and quality and RIN value measurement via HSRNA screentape assay or RNA screentape assay on the Agilent 4200 Tapestation system. Library preparation employed the Smart-3seq method on an Agilent Bravo Automated Liquid Handling Platform, using 100 ng input per sample. For lower-concentration samples, a pre-bead clean-up (poly A RNA capturing) with SPRI beads was performed. Indexes utilized were NEBNext Multiplex oligos for Illumina (E6440L) with 12 PCR cycles. Bead clean-up was done twice at a 0.8x ratio. Post-library preparation QC included concentration measurement using Qubit 1X dsDNA HS Assay Kit and BioTek Synergy H1 Multimode Reader, with molarity assessment via HSD5000 or D5000 screentape assay on the Agilent 4200 Tapestation system. Libraries were diluted to 4 nM and pooled. Superpool concentration and molarity verification employed the Qubit 1X dsDNA HS Assay Kit, Qubit 4 Fluorometer, and HSD5000 screentape assay.