title: RNA-Seq analysis of subcutaneous patient-derived neuroblastoma xenografts (LU-NB-3R): Comparison of BAY390, A967079, and vehicle controls
design: RNA-Seq analysis of subcutaneous patient-derived neuroblastoma xenografts (LU-NB-3R): Comparison of BAY390, A967079, and vehicle controls
library_construction_protocol: Treatment was administered for 21 days, after which mice were euthanized, tumor pieces were resected, cut to 3x3 mm pieces on ice and placed in RNA later-ICE transition solution (AM7030, Invitrogen) overnight.  Neuroblastoma in vivo model of LU-NB-3R was obtained via subcutaneous injection of human high-risk, MYCN-amplified neuroblastoma organoids in female NSG mice. When tumors reached 150 mm3, mice were randomized into treatment groups: control (n=8), A967079 (n=6), and BAY390 (n=7). All drugs were administered via oral gavage at the following concentrations: A967079 (300 mg/kg, once daily), BAY 390 (90 mg/kg, once daily) or vehicle for the control group. Treatment was administered for 21 days, after which mice were euthanized. All mice tolerated the treatment. Total RNA was extracted using the Tissuelyzer (85600, QIAGEN) and the AllPrep DNA/RNA Mini kit (80204, QIAGEN) according to the manufacturerâ€™s instructions. Quality was assessd by using the Agilent RNA ScreenTape Assay (G2991-90020 Rev.B) on the TapeStation 4200 System (Agilent). Concentration was measured with the QuantIT RNA High Sensitivity Assay Kit (Q33140, Thermo Fisher Scientific) using the Qubit Flex Fluorometer (Q33327, Invitrogen, Thermo Fisher Scientific).  Library preparation was performed according to the Illumina Stranded mRNA pep Ligation reference Guide (Document # 1000000124518 v03) with the change to 12 PCR cycles at the DNA fragment enrichment. Reagents used were the Illumina Standed MRNA Prep, Ligation (2004053, Illumina). Equipment used was the KingFisher FLEX (18-5400620, ThermoFisher) for automatized cleanup steps. Incubations and PCR were performed on the Eppendorf Mastercycler X50s (6311000010, Eppendorf). Library quality was measured according to the Agilent D5000 ScreenTape System Quick Guide, Part Number: G2964-90050 Rev. B, Edition 09/2015 using the Tape Station 4200 (Agilent) and D5000 ScreenTape (5067- 5588) with D5000 Reagents (5067- 558, Agilent). The concentration was measured using the Qubit Flex (Q33327, Invitrogen, Thermo Fisher Scientific) with the QuantIT 1X dsDNA HS Assay Kit (Q33232, Thermo Fisher Scientific) according to the Qubit Flex Fluorometer Quick Reference No. MAN0018187 Rev. A.0 protocol. 