title: Illumina HiSeq 2500 paired end sequencing; Analysis of HER3-EGFR score among pretreatment triple-negative breast cancer resection samples from the Georgia State University/University of Nottingham RNA-sequencing study (ICART1-2 cohort)
design: Analysis of HER3-EGFR score among pretreatment triple-negative breast cancer resection samples from the Georgia State University/University of Nottingham RNA-sequencing study (ICART1-2 cohort)
library_construction_protocol: Pre-marked tumor areas in n=60 formalin-fixed paraffin-embedded (FFPE) primary breast tumor resection samples obtained prior to treatment from unique triple-negative breast cancer patients were obtained from the Nottingham Primary Breast Carcinoma series, selected based on the presence of sufficient tissue quantity for the analysis. The FFPE samples were deparaffinized with xylene, after which RNA was extracted using the Omega Mag-Bind XP FFPE RNA isolation kit (Omega, M2595-01) and the KingFisher Flex magnetic particle separator (ThermoFisher). Integrity was evaluated using the Agilent 2200 TapeStation instrument (Agilent Technologies, Santa Clara, CA). First-strand cDNA synthesis using random hexamers and reverse transcriptase was performed on ~100 ng RNA (25°C for 10 min, 42°C for 15 min, and 70°C for 15 min). Second-strand synthesis and RNA sequencing libraries were prepared per the manufacturer's instructions using the TruSeq RNA Access library kit (Illumina, Inc., San Diego, CA, USA). RNA templates were removed and a second replacement strand was created by incorporating dUTP to generate double-stranded cDNA. Clean up was performed using Mag-Bind RxnPure Plus magnetic beads (Omega Bio-tek, Norcross, GA). cDNA 3' ends were adenylated, and indexing adaptors were ligated. DNA fragments with adapter molecules on both ends were enriched using PCR (15 cycles of 98°C for 10 sec, 60°C for 30 sec, and 72°C for 30 sec). The Agilent 2200 TapeStation and  QuantiFluor dsDNA System (Promega, Madiosn, WI) were used for library qualification and quantitation, respectively. 200 ng of each DNA library was combined to create a 4-plex pool of libraries, which were then mixed with capture probes specific to regions of interest, followed by hybridization (18 cycles of 1 min incubation, starting at 94°C, and then lowering by 2°C/cycle). Probes hybridized to the target regions were captured using streptavidin-coated magnetic beads. Enriched libraries were eluted and subjected to a second hybridization, then they were amplified by 10 cycles of PCR (98°C for 10 sec, 60°C for 30 sec, and 72°C for 30 sec). RxnPure magnetic beads were then used for clean-up. Final libraries were validated with the Agilent High Sensitivity D1000 ScreenTape on an Agilent 2200 Tapestation instrument.
attributes.tag: Experimental Factor: HER3-EGFR status
attributes.value: low