Mus musculus
Protocols: Mice were humanely culled by CO2 asphyxiation. Tumours were immediately removed and cut in half along the largest dimension. One half was preserved to be used for immunohistochemistry (IHC). The remaining half was dissected into ~1mm3 fragments, from which three were preserved in RNA later and frozen at -70°c after 30 minutes. For the xenograft model of A375 derived tumours in NSG mice, cells were cultured in MEM, until cells reached 100% confluence. Cells were detached with trypsin, centrifuged at 180 rcf for 5 minutes and resuspended in PBS at a concentration of 10 million cells/mL. 1 million cells were injected subcutaneously into the right flank of NSG mice. For treatment of NSG and C57BL/6 mice, CB-839 was given twice daily dose of 200mg/kg (10uL/g) via oral gavage, in a vehicle of 25% (w/v) hydroxypropyl-β-cyclodextrin in 10 mmol/L citrate (pH 2). PLX4032 (Adooq Bioscience) was given once daily via an intraperitoneal injection of 10mg/kg (4uL/g) in a vehicle of 4% DMSO, 4% TWEEN80 and 92% PBS.Subsequently to transplantation of cells, all mice were weighed and had their tumours measured three times each week. The endpoint for tumour growth was pre-defined as a maximal dimension of 15mm. All in vivo experiments were carried out in a dedicated barriered facility under the Animal (Scientific Procedures) act 1989. Tumour samples in RNAlater were thawed immediately before RNA extraction. Fragments were weighed and a mass of 200-300 tissue was used per sample from the available fragments. RNA extraction was performed using an RNeasy mini kit (Qiagen, 74106) with a RNase-Free DNase Set (Qiagen, 79254) and QIAshredder columns (Qiagen, 79656).Quality control of all RNA samples was performed using High Sensitivity RNA screen tape (Agilent, 5067-5579) on an Agilent Tapestation 2200. All samples had an RNA integrity number (RIN) value >8 and so were processed. RNA concentrations were determined by Qubit Fluorometer using the Qubit RNA Broad Range assay (both Thermo Fisher). 1ug of total RNA was used as input. Libraries were prepared using Illumina Stranded mRNA(Illumina, 2021) with IDT for Illumina RNA UD Indexes (Illumina, 20027213) used to index libraries. Post library QC was then performed using High Sensitivity D1000 screentape (Agilent, 5067-5584) for library sizing and profiling and quantified using Qubit High Sensitivity DNA assay. Libraries were then pooled equimolar, to a final concentration of 4nM prior to sequencing.
sampling site
neoplasm
strain
NSG
disease
metastatic melanoma
ENA-FIRST-PUBLIC
2023-04-07
organism
Mus musculus
organism
Mus musculus
xenograft
A375 injected subcutaneously into flank until neoplasm formation
ENA-LAST-UPDATE
2023-04-07
sex
male
scientific_name
Mus musculus
common name
house mouse
organism part
lateral lumbar region of abdomen
developmental stage
adult
ENA-FIRST-PUBLIC
2023-04-07
ENA-LAST-UPDATE
2023-04-07