Illumina NovaSeq 6000 paired end sequencing; Mutant FUS triggers age-dependent synaptic impairment in presymptomatic ALS-FUS mice (RNA-seq)
Cortex tissue was isolated from 1 month and/or 6-month-old FusΔNLS/+ and control Fus+/+ mice. Paired total homogenate(200ul) and SNS sample was obtained from a single mouse per condition using filtration protocol as previously described, nuclei fraction was also collected during SNS preparation, prior to filtration step in the SNS isolation protocol. Briefly, frozen total and SNS samples were mixed with Qiazol reagent following the manufacturer's recommendations and incubated at RT for 5 min. Two hundred microliters of chloroform were added to the samples and mixed for 15s and then centrifuged for 15 min (12,000g, 4C). To the upper aqueous phase collected, five hundred microliters of isopropanol and 0.8ul of glycogen was added and incubated at RT for 15 minutes. The samples were centrifuged at 10,000rpm for 10 min. After centrifugation at 12,000g for 15 min, the isopropanol was removed and the pellet was washed with 1 ml of 70% ethanol and samples were centrifuged for 5 min at 7500g. Ethanol was discarded and the RNA pellet was air-dried and dissolved in nuclease free water and further purified using the RNeasy Mini Kit including the DNAse I digestion step. The concentration and the RIN values were determined by Bioanalyser. 150ng of total RNA were used for Poly A library preparation. Strand specific cDNA libraries were prepared.