GSM8124744: PANC-1 cells, negative control, bioreplicate 2; Homo sapiens; OTHER
PANC-1 cells were incubated with 100 μg/mL CHX at 37 °C for 2 min to terminate the movement of ribosomes along mRNAs. Cells were then wash twice with ice-cold PBS containing 100 μg/mL CHX to remove dead cells and cell debris, and scratched off in ice-cold PBS containing 100 μg/mL CHX to collect PDAC cells. After centrifugation, the cell pellets were resuspended in polysome lysis buffer (20 mM HEPES (pH 7.4), 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 100 μg/mL cycloheximide), incubated on ice for 10 min, triturated ten times through a 26-G needle, and centrifuged at 20000g for 10min at 4℃ to collect supernatants. To prepare ribosome-protected fragments (RPFs), RNase I (NEB, Ipswich, MA, USA) and DNase I (NEB, Ipswich, MA, USA) were added into cell lysates to digest the free RNAs and the nuclease digestion reaction was kept for 45 min at RT with gentle mixing. Isolation of RPFs with a size greater than 17nt was achieved by size-exclusion chromatography with MicroSpin S-400 HR columns (GE Healthcare, USA) and RNA Clean and Concentrator-25 kit (Zymo Research; R1017) following the instructions of the manufacturer. After removing contaminating rRNA, the recovered RPFs were then applied for library construction using NEBNext® Multiple Small RNA Library Prep Set for Illumina®