title: RNA-Seq of MIA PaCa-2 cells
design: Total RNA from MIA PaCa-2 cells with vehicle control extracted using the Qiagen total RNA extraction kit. 500-1000ng of extracted RNA was used for further processing and analysis. Strand-specific RNA-seq libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina following the manufacturers recommendations. Total RNA quality was assessed using an RNA 6000 Nano kit on an Agilent 2100 Bioanalyzer (Agilent Biotechnologies), and the concentration was measured using a Qubit RNA HS assay kit (Life Technologies). A 500ng aliquot of total RNA was depleted of rRNA using a Human/Mouse/Rat RNAse-H based Depletion kit from NEB (New England Biolabs). Following rRNA removal, mRNA was fragmented and then used for first- and second-strand cDNA synthesis with random hexamer primers. Double-stranded cDNA fragments underwent end repair, poly(A) tailing and the ligation of two unique adapters (Integrated DNA Technologies, Coralville, IA). The adaptor-ligated cDNA was amplified by limited-cycle PCR. Library quality was analyzed with TapeStation High-Sensitivity D1000 ScreenTape (Agilent Biotechnologies) and quantified by KAPA qPCR (KAPA BioSystems). Libraries were pooled and sequenced at 2x150-bp read lengths on the Illumina HiSeq 4000 platform. 151-bp paired-end RNA-seq reads generated for each sample (were aligned to the human and mouse genome using Rsu-bread (>90% of ~25x106 unique reads/sample mapped) and translated to expression counts via feature Counts, followed by analysis via the standard edgeR pipeline to DEGs, visualization with Volcano plots (ggplot2), and interpretation by GSEAs; in particular, enrichment of a hallmarks gene set was analyzed using GSEA.
library_name: VC_1_R1