RNA-seq analysis of co-treatment with gemcitabine and N-Methyl-(5-pyrid-3- ylthien-2-yl)methylamine in PANC-1 cells
RNA-seq analysis of co-treatment with gemcitabine and N-Methyl-(5-pyrid-3- ylthien-2-yl)methylamine in PANC-1 cells
Treated PANC1 cells were harvested by trypsinisation (0.05% trypsin, 5 minutes at 37°C) at the endpoint of treatment (96 hours post-co-administration of gemcitabine and BD B10). Cells were pelleted by centrifugation (1000 g, 5 minutes, room temperature), washed three times with 1x PBS, and stored at -80°C.  PANC1 cells were cultured in DMEM with 10% heat-inactivated FBS and 1% penicillin-streptomycin at 37°C, 5% CO₂. Cells were passaged 3–4 days (1:3 ratio, 0.05% trypsin for 5 minutes at 37°C). PANC-1 cells were seeded at 5,000 cells/well in 96-well plates, pre-treated with 10 µM BD B10 for 24 h, then the medium was replaced with fresh medium containing 0.1 µM gemcitabine + 10 µM BD B10 for an additional 96 h incubation at 37°C, 5% CO₂. Control conditions included: (1) untreated, (2) BD B10 alone (10 µM), and (3) gemcitabine alone (0.1 µM). Total RNA was extracted from snap-frozen Panc1 cell pellets using the RNeasy Plus Mini Kit (74136, Qiagen) according to the manufacturer's instructions. RNA quantity and purity were assessed using a NanoDrop ND-1000 spectrophotometer.  Stranded, polyA-enriched mRNA libraries were constructed from 1 µg total RNA using the Illumina TruSeq Stranded mRNA Library Prep Kit following the manufacturer's protocol.   