Illumina HiSeq 2500 paired end sequencing; Mutant FUS triggers age-dependent synaptic impairment in presymptomatic ALS-FUS mice (RNA-seq)
Total lysate and synaptoneurosomes isolated from cortex tissue of 1-month old C57Bl/6 mice were UV crosslinked (100 mJ/cm2 for 2 cycles) using UV Stratalinker 2400 (Stratagene) and stored at -80°C until use. For the total sample, cortex tissue was dissociated using a cell strainer of pore size 100 μm before crosslinking. We used cortex from 200 mouse to prepare SNS and two mice for the total cortex sample. Cross-Linking Immunoprecipitation and high-throughput sequencing (CLIP-seq): We used a mouse monoclonal antibody specific for the C-terminus of FUS (Santa Cruz) to pull down FUS associated RNAs using magnetic beads. After immunoprecipitation, FUS-RNA complexes were treated with MNAse in mild conditions and the 5' end of RNAs were radiolabeled with P32-gamma ATP. Samples run on SDS-gel (10% Bis Tris) were transferred to nitrocellulose membrane and visualized using FLAphosphorimager. CLIP libraries were prepared using the Clontech SMRTer Stranded total RNA-Seq Kit-Pico Input Mammalian Total RNA according to manufacturer's instructions.