library_construction_protocol: Intracranial tumor-bearing mice were euthanized and then perfused via transcardial injection of 10 mL of cold PBS. Whole brains were harvested, and visible tumor tissues were excised and stored in RNAlater (Invitrogen, AM7024) at 4°C for up to one week before RNA extraction. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, 74106) and the RNase-Free DNase Set (Qiagen, 79254), following the manufacturer's protocol. Because of their low RIN scores, we chose ribosomal depletion for mRNA enrichment. The subsequent library preparation and sequencing were performed by the UCSF Genomics CoLab. Starting material of 500 ng of total RNA was used according to vendor instructions with Universal plus mRNA with Nu Quant (TECAN, 0520), with noted changes to the protocol as follows. First, the Poly(A) Selection step was omitted. Second, we started with RNA fragmentation and used QIAseq FastSelect −rRNA/Globin Kit (Qiagen, 335377) to remove rRNA as follows: 0.1 µL of FastSelect rRNA and 1 µL of 1X Fragmentation Buffer was added to 10 µL of total RNA, with the addition of 10 µL of 2X Fragmentation Buffer. PCR program was used as follows: 94 ℃/3min, 75 ℃/2min, 70 ℃/2min, 65 ℃/2min, 60 ℃/2min, 55 ℃/2min, 37 ℃/5min, 25 ℃/5min, 10 ℃/hold. After the rRNA depletion step, Universal plus mRNA with Nu Quant protocol was followed, starting with the first strand cDNA synthesis step but omitting the steps of AnyDeplete and NuQuant. After final library PCR amplification of 15 cycles and bead clean-up, individual libraries were pooled equally by volume, and quantified on Fragment Analyzer (Agilent, DNF-474). The quantified library pool was diluted to 1 nM and sequenced on MiniSeq (Illumina, FC-420-1001) to check for the quality of reads. Finally, individual libraries were normalized according to MiniSeq output reads, specifically by % protein-coding genes, and were sequenced on one lane of NovaSeq6000 SP PE150 (Illumina, 20028400) at UCSF Center for Advanced Technology (CAT).