title: BAG6KO2
organism: Mus musculus
description: Protocols: EVs were collected in cell culture for 48h in protein-free CD293 medium and isolated by (ultra-) centrifugation. The (ultra-)centrifugation protocol included consecutive pre-centrifugation steps at 300 x g (10 min), 2,000 x g (10 min) and 3,500 x g (20 min) for clearance of cells and cellular debris before ultracentrifugation at 100,000 x g (90 min) using rotor Type 45Ti. EVs were washed two times by resuspension in PBS, with the final run using TLA-45 rotor. B-16V (CVCL_1950)cells were maintained according to the DSMZ guidelines in RPMI (Gibco, USA), supplemented with 0.5% penicillin-streptomycin (Gibco, USA), and 10% fetal bovine serum (Gibco, USA). Cells were kept under standard culturing conditions with 5% CO2, 37 degrees centigrade. EV pellet was resuspended in lysis buffer and RNA was isolated using the RNeasy Mini Kit according to the manufacturer's protocol recommended for processing animal cell lysates. TruSeq Stranded mRNA Library Preparation Kit