title: GSM7865626: PtPM Tumor - Rep 8; Mus musculus; RNA-Seq
library_construction_protocol: Tumors were isolated from euthanized mice and validated for EGFP fluorescence under fluorescence stereomicroscope. Tumors were quickly placed within 250-500 μL of RLT buffer supplemented with B-mercaptoethanol into 2mL tube with ceramic beads (MP, 6910500). Tumor samples were lysed on a Fisher Bead Mill 24 using manufacturer recommended settings. Lysates were passed through a Qiashredder (Qiagen, 79656) and RNA isolated using the Qiagen RNeasy kit (Qiagen, 74106) with manufacturer recommended protocols. Organoids were dissociated as above and resuspended in 300 μL of RLT buffer supplemented with B-mercaptoethanol and spun through a Qiashredder. Qiagen RNeasy kit was performed to isolate RNA using manufacturer recommended protocols. After RiboGreen quantification and quality control by Agilent BioAnalyzer, 500 ng of total RNA with RIN values of 8.3-10 underwent polyA selection and TruSeq library preparation according to instructions provided by Illumina (TruSeq Stranded mRNA LT Kit, catalog # RS-122-2102), with 8 cycles of PCR. Samples were barcoded and run on a NovaSeq 6000 in a PE100 run, using the NovaSeq 6000 SX Reagent Kit (Illumina). An average of 24 million paired reads was generated per sample. Ribosomal reads represented 0.4-1.5% of the total reads generated and the percent of mRNA bases averaged 86%.