RNA-Seq analysis

CLB-BAR and CLB-GE cells were seeded on 15 cm³ dishes in triplicate prior to treatment with 50 nM BAY 1895344 for 24 and 48 h. Control samples were harvested after 48 h. RNA was extracted and RNA-Seq performed by Novogene UK.

Rosa26_Alkal2Tg/0;TH-MYCNTg/0 and Alk-F1178SKI/0;TH-MYCNTg/0 on a 129×1/SvJ background (JAX stock #000691) (n = 4 per genotype) were screened by ultrasound, 2–3 times a week from approximately 35 days of age. Tumours were followed until they reached a size of 8–12 mm in average diameter after which a 3D scan of the tumour was made and treatment started with BAY 1895344 25 mg/kg b.i.d. P.O. for 3 days. A 3D scan was repeated at day 3, the mice were sacrificed and tumour extracted and incubated in RNAlater (Thermo Fisher #AM7020). RNA was extracted using the ReliaPrep™ RNA Miniprep Systems (Promega #6111) according to the manufacturer’s protocol. The purified RNA was sent to Novogene UK for library preparation and sequencing. Images and 3D measurements were analysed in VevoLab (Fujifilm VisualSonics, Toronto, Canada).

RNA-Seq paired-end reads (read length 150 base pairs) were aligned to the GRCh38 (human cell line data) or GRCm38 (mice data) reference genome using hisat2⁶⁷. The average alignment efficiency was 90.9% and 90.3% for human and mice data, respectively. Genes were annotated using GENCODE 29 (human) or M22 (mouse)⁶⁸ and quantified using HTSeq⁶⁹. Only coding genes were used for further analysis. Differential gene expression was determined using DESeq2⁷⁰. Genes were considered differentially expressed if their absolute log2 fold change values were above 1.5 at FDR-adjusted p values (Padj) below 0.01, considering a hyperbolic threshold given by:

With Padj_TH and log2FC_TH representing the adjusted p value (0.01) and log2 fold change (1.5) thresholds, respectively, and c the curve parameter (here c = 0.5).