Commercially obtained N2A mouse neuroblastoma cells (ATCC, CCL-131), which were authenticated by ATCC and free of mycoplasma, were cultured in Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (DMEM/F-12, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (VWR). Cells (0.3 × 106/6-well plate) were transiently transfected with 60 pmol of Ambion Silencer Negative Control #1 siRNA (Thermo Fisher Scientific) or Upf1 siRNA (Additional file 3 : Table S2) using the TransIT-X2 Dynamic Delivery System (Mirus Bio). For p-UPF1 RIP-seq, N2A cells were treated with 200 nM okadaic acid (Sigma) for 2 h prior to cell lysis and immunoprecipitation.
Cerebral cortical tissues were removed from WT or Fmr1-KO P1 pups and dissociated using papain protease (Worthington) and mechanical trituration. Dissociated cells were filtered using 40 μm cell strainers (BD Falcon) to remove blood vessels and tissue clumps, then immune-depleted of ACSA2+ astrocytes [41 (link)] and myelin using anti-ACSA-2 and anti-myelin microbeads (Miltenyi Biotech), respectively, and were then plated on 5 ng/ml laminin/poly-L lysine substrate (Thermo Fisher Scientific) at 4.2 × 104 cells/cm2 and cultured for 2 days in Neurobasal/B27 Plus Medium (Thermo Fisher Scientific) supplemented with 1× penicillin-streptomycin solution (Thermo Fisher Scientific).
Cerebral cortical tissues were removed from WT or Fmr1-KO P1 pups and dissociated using papain protease (Worthington) and mechanical trituration. Dissociated cells were filtered using 40 μm cell strainers (BD Falcon) to remove blood vessels and tissue clumps, then immune-depleted of ACSA2+ astrocytes [41 (link)] and myelin using anti-ACSA-2 and anti-myelin microbeads (Miltenyi Biotech), respectively, and were then plated on 5 ng/ml laminin/poly-L lysine substrate (Thermo Fisher Scientific) at 4.2 × 104 cells/cm2 and cultured for 2 days in Neurobasal/B27 Plus Medium (Thermo Fisher Scientific) supplemented with 1× penicillin-streptomycin solution (Thermo Fisher Scientific).