title: RNA seq of C57B6 mice KPC tumor treated with IKE and 10A3
design: RNA seq of C57B6 mice KPC tumor treated with IKE and 10A3
library_construction_protocol: we used high-throughput next-generation sequencing technology to compare transcriptomics between each samples that were harvested from the mouse xenografts  Total RNA was extracted and purified from cultured cells using the RNeasy Plus Mini Kit (QIAGEN, 74136) according to the manufacturer's instructions.  An Agilent 2100 Bioanalyzer system and RNA Nano chip kit (5067-1511) were used for RNA quality measurement. An Illumina TruSeq Stranded mRNA Library prep kit (20020594) was used to generate the RNA libraries. The first step in the workflow involved purifying the poly-A containing mRNA molecules using oligo-dT attached magnetic beads. Following purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers. Second-strand cDNA synthesis followed, using DNA polymerase I and RNase H. The cDNA fragments then went through an end repair process, the addition of a single “A” base, and then ligation of the adapters. The products were then purified and enriched with PCR to create the final cDNA library. AMPure XP beads (Beckman Coulter, A63881) purifications were performed two times before the assessment took place. The library quantity was measured using a picogreen method. A Quant-iT PicoGreen dsDNA Assay kit (Invitrogen, P7589) and PerkinElmer plate reader (PerkinElmer Victor X3, 2030 Multilabel Reader) were used in the assessment. The library quality was verified on an Agilent 2100 Bioanalyzer instrument using an Agilent DNA 1000 kit (5067-1504). All libraries must have met the QC requirements before we moved forward with sequencing.