Cell culture and transfection

U-2 OS (American Type Culture Collection HTB-96) and HEK293T (American Type Culture Collection CRL-1573) cells were grown in DMEM supplemented with 10% (v/v) FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma–Aldrich). To generate polyclonal stable cell lines, U-2 OS cells (U2PA stably expressing stdMCP-Halotag and stdPCP-stdGFP¹⁵ for splicing reporter, U2PA stably expressing stdMCP-Halotag and scFv-sfGFP²¹ for Suntag translation reporter) were transfected with linearized reporter constructs by nucleofection (Lonza nucleofector 2b, VACA-1003), followed by 200 µg/ml hygromycin selection for 20 days. HEK293T cells were transfected by TransIT®-LT1 Transfection Reagent (Mirus Bio). ALS patient-derived fibroblast cell lines (C9-2, C9-5, C9-6) and healthy control fibroblast cell lines (Ctrl-1, Ctrl-2) are gifts from Dr. John Ravits²⁵ were grown in DMEM supplemented with 20% (v/v) FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM Glutamine and 1% (v/v) nonessential amino acids. Patient lymphoblast cells were obtained from Coriell Institute and grown in RPMI1640 supplemented with 10% (v/v) FBS, 2 mM L-Glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cell line information is listed in Supplementary Table ¹.

Lipofectamine^® RNAiMAX (Invitrogen) was used to transfect siRNAs and ASOs. ON-TARGETplus pooled siRNAs of NXF1 (GE Dharmacon, L-013680-01-0005), NXT1 (L-017194-00-0005), DDX39B (L-003805-00-0005), ENY2 (L-018808-01-0005), THOC1 (M-019911-01-0005), THOC7 (M-014575-01-0005), and nontargeting control (D-001810-10-05) were transfected at 25 nM and cells were harvested after 3 days. Nontargeting ASO (CCTTCCCTGAAGGTTCCTCC) and the C9ORF72-targeting ASO (GCCTTACTCTAGGACCAAGA)²⁵ were transfected at 25 nM, and cells were collected after 2 days.