GSM8214600: PANC-1 cells, YBX1 IP-rep2; Homo sapiens; RIP-Seq
PANC-1 cells were pretreated with CAF-deirved Evs. Pretreated cells were UV-crosslinked (at 254nm, 400 mJ/cm2) and lysed with RIP lysis buffer. Lysed pellets were sonicated and treated with RNaseI (Life Technologies) Turbo DNase (Life Technologies). Then magnetic beads coated with 1 μg of YBX1 antibody were added into cell lysate and incubated overnight at 4 °C. The precipitated RNA was released with proteinase K and extracted 2 times with phenol chloroform isoamyl alcohol, followed by ethanol precipitation with a glycogen coprecipitant, 80% ethanol wash and final resuspension in TE. Illumina sequencing libraries were generated with NEBNext® Ultra™ DNA Library Prep Kit (New England Biolabs) by following the manufacturer's manual. The library quality was determined by using Agilent 2100 Bioanalyzer (Agilent), and then, subjected to high-throughput 150 base paired-end sequencing on Illumina Novaseq sequencer according to the manufacturer's recommended protocol.