characteristics: ERC000011
characteristics: ENA-CHECKLIST
characteristics: 2022-11-01T00:15:41Z
characteristics: ENA-FIRST-PUBLIC
characteristics: 2022-11-01T00:15:41Z
characteristics: ENA-LAST-UPDATE
characteristics: SAMEA111342872
characteristics: External Id
characteristics: Department of Surgery, UT Southwestern Medical Center
characteristics: INSDC center name
characteristics: 2022-11-01T00:15:41Z
characteristics: INSDC first public
characteristics: 2022-11-01T00:15:41Z
characteristics: INSDC last update
characteristics: public
characteristics: INSDC status
characteristics: ERS13440430
characteristics: SRA accession
characteristics: E-MTAB-12168_3:Sample 5
characteristics: Submitter Id
characteristics: 65
characteristics: year
characteristics: age
characteristics: ArrayExpress
characteristics: broker name
characteristics: MIA Paca-2
characteristics: cell_line
characteristics: epithelial cell
characteristics: cell_type
characteristics: human
characteristics: common name
characteristics: Protocols: MIA PaCa-2 (a human PDAC cell line) was used in the experiment. To generate G12Ci-resistant cell lines, we cultured KRAS-G12C-mutant cells with increasing concentrations of G12Ci MRTX849 (100 nM-50,000 nM) for 6 months. The parental and MRTX849-resistant MIA PaCa-2 cells were cultured in the absence or presence of MRTX849 (1 uM, 4h). 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.
characteristics: description
characteristics: pancreatic ductal adenocarcinoma
characteristics: disease
characteristics: wild type genotype
characteristics: genotype
characteristics: Homo sapiens
characteristics: organism
characteristics: Homo sapiens
characteristics: organism
characteristics: pancreas
characteristics: organism part
characteristics: Homo sapiens
characteristics: scientific_name
characteristics: male
characteristics: sex
characteristics: Sample 5
characteristics: title