Protein a beads

Total RNA (>300 µg) of samples were chemically fragmented into ~100-nucleotide-long fragments by 5 min incubation at 94 °C in fragmentation buffer (10 mM ZnCl2, 10 mM Tris-HCl, pH 7). The fragmentation reaction was stopped with 0.05 M EDTA, followed by standard ethanol precipitation. Samples were resuspended in H₂O at ~1 mg ml/1 concentration and subjected to m⁶A-seq. Fragmented RNA was incubated for 2 h at 4 °C with 5 mg of affinity-purified anti-m⁶A polyclonal antibody (Synaptic Systems cat. no. 202 003) in IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.4). The mixture was then immunoprecipitated with protein-A beads (Repligen) at 4 °C for an additional 2 h. After extensive washing, bound RNA was eluted from the beads with 0.5 mg ml/1 N⁶-methyladenosine (Sigma-Aldrich) in IPP buffer, and ethanol precipitated. RNA was resuspended in H₂O and used for library generation with the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs). Sequencing was carried out on Illumina HiSeq2500 according to the manufacturer’s instructions, using 10 pM template per sample for cluster generation, and sequencing kit V2 (Illumina). These raw data are available at NCBI SRA series PRJNA701370 (public release date set to 2021-08-01).

Simple cloning was performed to produce the ICAM‐1 protein. Mouse or human ICAM‐1 extracellular domain was cloned into a mouse (Invivogen, pfuse‐mchg1) or human IgG expression vector (Invivogen, pfuse‐hg40fc1). The plasmid was transfected into Expi293F cells using the ExpiFectamine 293 Transfection kit (Gibco, A14524). The protein was then purified from the supernatant using protein A beads (REPLIGEN, 10‐2500‐03) and an IgG Elution Buffer (Thermo Fisher Scientific, 21004).

Male and female mice were deeply anesthetized with isofluorane vapor before rapid decapitation. Whole brains were quickly transferred to a solution of 1% Triton X-100 buffer with protease inhibitors and homogenized via a mortar and pestle. The homogenate was then spun at 90,000 g for 30 min at 4 C to remove debris. The resulting supernatant was then collected and pre-cleared with 0.1% Triton X-100 washed protein-A beads (RepliGen Cat # 10-1003-01) for 1 h at 4 C. A small portion of pre-cleared supernatant was saved for input, and the remaining supernatant was then incubated with 2 µg of Rabbit anti-V5-tag antibody (Cell Signaling Technology Cat# 13202) or 2 µg of control rabbit IgG (Jackson ImmunoResearch Cat# 309-005-008) and 30 µL of protein-A beads for 4 h rotating head over tail at 4 C. The antibody bound beads were then washed three times with 0.1% Triton X-100 and pelleted to isolate the protein bound beads. The supernatant was then removed and the bead bound proteins were eluted with 100 µL of 1x sample buffer and boiled for 5 min at 100 C.