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Protocol publication

[…] were isolated as described above. For these studies it was necessary to pool large numbers of hippocampi and therefore biological replicates were not attempted. Upf1 (Abcam ab109363) was immunoprecipitated from samples using Protein-G-conjugated Dynabeads (Thermo Fisher Scientific) and Upf1-bound RNA was extracted using TRIzol (Thermo Fisher Scientific). Ribosomal RNA was removed from the co-precipitated RNA using the Ribo-Zero Magnetic Kit (Illumina) and the RNA library was constructed using the ScriptSeqTM v2 RNA-Seq Library Preparation Kit (Illumina) according to the manufacturer’s instructions. 3′Adapter sequences were removed and the quality of sequenced reads were first checked using FASTqc software. Reads were then mapped to the mouse genome (Dec. 2011 (GRCm38/mm10) assembly) using Bowtie, analysed using TopHat and Cufflinks free open source software. FPKM cut-off for gene presence was ≥1., Transcripts identified by RIP-seq were categorised based on their regulation. Unregulated transcripts were sorted into 3 groups: those present in both the cytoplasm and synaptoneurosomes, those only found in synaptoneurosomes and those found exclusively in the cytoplasm. Regulated genes were divided into 4 groups: Genes expressed exclusively in control SNs or with a log2-fold decrease ≤−1.5, genes expressed exclusively in SE SNs or with a log2-fold increase ≥−1.5, genes expressed exclusively in control cytoplasm or with a log2-fold decrease ≤−1.5, genes expressed exclusively in SE cytoplasm or with a log2-fold incre […]

Pipeline specifications

Software tools FastQC, TopHat, Cufflinks
Organisms Mus musculus, Homo sapiens
Diseases Tooth Demineralization, Brain Diseases, Brain Diseases, Seizures, Neurologic Manifestations, Seizures, Neurologic Manifestations