title: GSM9386807: AKP_tumors; Mus musculus; RNA-Seq
library_construction_protocol: Mouse tumor samples harvested after termination were washed in PBS and mechanically minced into small fragments using sterile blades. Subsequently, tumor fragments were enzymatically incubated in a digestion buffer containing collagenase II at 2.5 mg/ml (Sigma-Aldrich, C6885-1G), collagenase IV at 2.5 mg/ml (Sigma-Aldrich, C5138-1G), and DNase I at 0.5 mg/ml (Roche; #11284932001) for 40 minutes at 37°C on a gently rotating platform to enhance enzymatic activity. Following digestion, the tumor suspension was filtered through a 100 µm cell strainer. FBS was immediately added to the collected fraction to neutralize enzymatic activity. The samples were centrifuged at 320 × g for 5 minutes, after which the supernatant was discarded. Cell pellets were washed three times with sterile PBS, centrifuging at 320 × g for 5 minutes between washes. After the final wash, cells were resuspended in 200 µL sterile PBS. RNA was then extracted using the High Pure RNA Isolation Kit (Roche; #11828665001), following the manufacturer's protocol. Purified RNA samples were subjected to bulk RNA sequencing using the Mercurius BRB-seq platform from Alithea Genomics (reference), according to the manufacturer's instructions. Sequencing was performed on an Illumina NovaSeq instrument (Aviati platform) with a sequencing depth of 5 million reads per sample, using a single-end 150 bp (SE150) configuration at the Genomic Technologies Facility (GTF), University of Lausanne (UNIL), Lausanne, Switzerland