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Accession IconSRP071321

Extensive cryptic splicing upon loss of RBM17 and TDP43 in neurodegeneration models

Organism Icon Mus musculus
Sample Icon 29 Downloadable Samples
Technology Badge IconIllumina HiSeq 2500

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Translating ribosome affinity purification technology was used to isolate mRNAs from cerebellar Purkinje neurons from control (Pcp2-BacTrap; Rbm17 f/+) and mutant (Pcp2-BacTRAP; Pcp2-Cre; Rbm17 f/-) mice. Overall design: RNA isolation was performed when animals were four-weeks-old (n=3 animals per genotype). Using NuGEN Ovation RNA-Seq System v2, purified double-stranded cDNA was generated from 10 ng of total RNA and amplified using both 3' poly (A) selection and random priming. 2 µg of each sample was sheared using the Covaris S2 focused-ultrasonicator following the manufacturer's protocol to obtain a final library with insert size of 400 bp. The sheared samples were quantified using the NanoDrop ND-1000 spectrophotometer and Invitrogen Qubit 2.0 DNA quantitation assay. The fragment sizes were confirmed on the Agilent Bioanalyzer to verify proper shearing. A double-stranded DNA library was produced using Illumina TruSeq DNA library preparation system and the sequencing was run on a HiSeq 2500 system.
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