Tissuelyser equipment

Approximately 100mg of motor cortex from 4 sporadic ALS and 4 C9orf72‐ALS cases was lysed in 10× RIPA (50 mM Tris–HCl pH 7.8, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP40; supplemented with protease inhibitors and EDTA) volume using TissueLyser equipment (Qiagen). Lysates were incubated on ice 20 min followed by centrifugation at 20,000 g for 20 min at 4°C. Supernatant was taken as “RIPA fraction”, and pellets were resuspended in RIPA and SDS (final concentration of 2%). 3 sporadic ALS and 3 C9orf72‐ALS samples were subsequently used as they were sufficiently concentrated to load 100 ug of proteins onto SDS–PAGE gels.

Frozen tissues were placed in 2 mL eppendorf tubes containing 1 mL Trizol (Thermo Fisher Scientific) and mechanically disgregated using the bead-based TissueLyser equipment (Qiagen) by shaking for 30 s at 30 Hz in presence of one bead per tube. After brief centrifugation to remove tissue debris (3 min at 2000 RPM at 4°C), 300 μL chloroform were added, samples were shaken and centrifuged for 10 min at 10000 RPM at 4°C. The chloroform phase was collected, mixed with 600 μL isopropanol and centrifuged again as before. The pellet obtained was washed twice with 70% ethanol and finally resuspended in 50 μL PCR-grade water. For retrotranscription, 1 μg RNA was used per sample using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qPCR were performed on a LightCycler 480 machine (Roche) with SYBR Green-based detection (Roche). The primer sequences are shown in the Key Resources Table.