C57bl 6j mice

ID8, ID8-c-MYC, and ID8-KRAS cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, NY, USA) containing 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 g/ml amphotericin B. C57BL/6J mice were chosen because ID8 was established from C57BL/6J mice [12 (link)]. Mice were purchased from Japan SLC, Inc. ID8, ID8-KRAS, and ID8-c-MYC cells (2× 10⁶) suspended in 1000 μl of DMEM were injected into the peritoneal cavities of 8-week-old female mice under anesthesia by isoflurane. ID8-KRAS mice were monitored every other day, and ID8 and ID8-c-MYC mice were monitored twice a week. Mice were sacrificed by isoflurane when their body weight (BW) reach 23 g after the inoculation. At the time of sacrifice, BW and ascites weights were assessed. Mice were sacrificed to minimize suffering when moribund behaviors were observed. Total number of mice used in this study was 82 as following: for observation of phenotype and sample collection, ID8 mice group: n = 10, ID8-c-MYC mice group: n = 10, ID8-KRAS mice group: n = 10, for time course analysis: n = 20, for Giemsa assay: n = 16 (n = 4 per group), for the assessment of leukocytes proportion by FACS: control mice: n = 4, ID8 mice: n = 6, and ID8-KRAS mice: n = 6. Animal studies were approved by the University of Tokyo Animal Committee.

m⁶A-MeRIP using low-input materials was performed based on a previously described protocol [55 (link)] with some modifications. Briefly, total RNA from about 2500 mouse GV oocytes from 5 weeks C57BL/6 J mice (Beijing Vital River Laboratory Animal Technology Co., Ltd) was first randomly fragmented to ~200 nt with RNA fragmentation reagents (AM8740, Thermo, USA), and then incubated with the protein A beads (10001D, Thermo, USA) coupled with anti-m⁶A polyclonal antibody (ABE572, Millipore, USA) in IPP buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% NP-40, 0.4 U/μl RNasin). After immunoprecipitation, the RNA reaction mixture was washed twice in 1 ml of IP buffer, twice in 1 ml of low-salt IP buffer (50 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% IGEPAL^® CA-630 (I8896, Sigma-Aldrich, Germany) in nuclease-free H₂O), and twice in 1 ml of high-salt IP buffer (500 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% IGEPAL CA-630 in nuclease-free H₂O) for 5 min each at 4 °C. After extensive washing, the m⁶A-enriched RNA fragments were eluted from the beads by proteinase K digestion followed by phenol-chloroform extraction and ethanol precipitation. The purified RNA was subjected to library construction using the SMARTer Stranded Total RNA-Seq Kit v2 (634413, Clontech, Japan) according to the manufacturer’s instructions. Sequencing was performed on an Illumina HiSeq X-ten sequencing system.

Five- to seven-week-old C57BL/6J mice (The Jackson Laboratory, RRID:IMSR_JAX:000664) were terminally anesthetized in a CO₂ chamber and then transcardially perfused with 1× DPBS (Gibco 14190-250; Thermo Fisher Scientific, Waltham, MA, USA). The cerebellum was extracted and transferred into chilled artificial cerebrospinal fluid (124 mM NaCl; Macron 7581-12, 2.5 mM KCl; Sigma-Aldrich P3911, 2.0 mM MgSO₄; Sigma-Aldrich M3409, 1.25 mM KH₂PO₄; Sigma-Aldrich P5655, 26 mM NaHCO₃; Sigma-Aldrich 93350, 10 mM glucose; Macron 4912-12). The cerebellum was sectioned into 300 µm coronal slices using a vibratome Ci 7000 smz (Campden Instruments; Lafayette, IN, USA). Cerebellum slices were submerged in Minimum Essential Media (MEM; Gibco 11095; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 15% fetal bovine serum (FBS; Gibco 26140079; Themo Fisher Scientific, Waltham, MA, USA), 100 mg/mL penicillin G, and 100 mg/mL streptomycin (Gibco 15140122; Thermo Fisher Scientific, Waltham, MA, USA) until use [72 (link)].