title: RNA-seq of irradiated intestinal organoid-derived subcutaneous heterotopic tumours and non-irradiated controls
design: RNA-seq of irradiated intestinal organoid-derived subcutaneous heterotopic tumours and non-irradiated controls
library_construction_protocol: Tumours were excised from radiation-treated mice at 4 hours, 24 hours, 3 days, and 7 days after the completion of each irradiation scheme (15Gy and 3x7Gy) as well as from corresponding untreated/non-irradiated mice and stored in RNAprotect tissue reagent (Qiagen) at -20°C prior to RNA extraction. The AKPT (villinCreER;Apcfl/fl;KrasG12D/+;Trp53fl/fl;TgfbrIfl/fl) organoids were maintained in Advanced Dulbecco's Modified Eagle Medium/F12 (Thermo Fisher) supplemented with penicillin/streptomycin, 10 mmol/L HEPES, Glutamax, 1x N2 supplement (Thermo Fisher), 1× B27 supplement (Thermo Fisher), 1.25 mmol/L N-acetyl-l-cysteine (Sigma-Aldrich), 50 ng/mL recombinant murine epidermal growth factor (Thermo Fisher) and 100 ng/mL noggin (PeproTech). Organoids were suspended in a phosphate-buffered saline and Matrigel (Corning) mixture (50:50). Organoids were then implanted into male C57BL/6 mice subcutaneously, monitored, and treated two weeks after implantation. Once the subcutaneous tumour volumes reached a volume of 150-200 mm3, mice were given either 15 Gy radiation as a single dose or 3 doses of 7Gy, using the Gulmay Medical RS320 irradiator (300kV, 10mA, 1.81Gy/min). For the fractionated irradiation, tumours received 7Gy of radiation three times, with 24 hours gap between each dose. Tumours were harvested after recieving the single dose of 15Gy or after the final dose of 7Gy at the previously described timepoints (4 hours, 24 hours, 3days, 7days).  was extracted using the RNeasy Micro Kit from Qiagen (74004) and DNase treatment was performed using the DNA-free kit from Life Technologies (AM1906). The starting RNA material was quantified using RiboGreen (Invitrogen) on the FLUOstar OPTIMA plate reader (BMG Labtech) and the size profile and integrity analysed on the TapeStation (RNA ScreenTape, Agilent). 3'RNAseq libraries were prepared using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD (Lexogen, Austria) and the UMI Second strand synthesis module for QuantSeq FWD (Lexogen, Austria) following the manufacturer's instructions. RNA input varying from 100-1000ng and 14-20 PCR cycles were used. Library quality and quantity were assessed using the TapeStation (Agilent) and Qubit Assay (ThermoFisher) respectively. Individual libraries were normalised and pooled accordingly to multiplex for sequencing.