Deep Sequencing Reveals Uncharted Isoform Heterogeneity of the Protein-Coding Transcriptome in Cerebral Ischemia Sunil Bhattarai 1 & Ahmed Aly 1 & Kristy Garcia 1 & Diandra Ruiz 1 & Fabrizio Pontarelli 1 & Ashutosh Dharap 1 Received: 12 March 2018 /Accepted: 22 May 2018 # Springer Science+Business Media, LLC, part of Springer Nature 2018 Abstract Gene expression in cerebral ischemia has been a subject of intense investigations for several years. Studies utilizing probe-based high-throughput methodologies such as microarrays have contributed significantly to our existing knowledge but lacked the capacity to dissect the transcriptome in detail. Genome-wide RNA-sequencing (RNA-seq) enables comprehensive examinations of transcriptomes for attributes such as strandedness, alternative splicing, alternative transcription start/stop sites, and sequence composition, thus providing a very detailed account of gene expression. Leveraging this capability, we conducted an in-depth, genome-wide evaluation of the protein-coding transcriptome of the adult mouse cortex after transient focal ischemia at 6, 12, or 24 h of reperfusion using RNA-seq. We identified a total of 1007 transcripts at 6 h, 1878 transcripts at 12 h, and 1618 transcripts at 24 h of reperfusion that were significantly altered as compared to sham controls. With isoform-level resolution, we identified 23 splice variants arising from 23 genes that were novel mRNA isoforms. For a subset of genes, we detected reperfusion time- point-dependent splice isoform switching, indicating an expression and/or functional switch for these genes. Finally, for 286 genes across all three reperfusion time-points, we discovered multiple, distinct, simultaneously expressed and differentially altered isoforms per gene that were generated via alternative transcription start/stop sites. Of these, 165 isoforms derived from 109 genes were novel mRNAs. Together, our data unravel the protein-coding transcriptome of the cerebral cortex at an unprec- edented depth to provide several new insights into the flexibility and complexity of stroke-related gene transcription and transcript organization. Keywords Mouse . Ischemia . Cerebral cortex . RNA-sequencing . Gene expression Introduction Stroke is one of the leading causes of death and long-term disability in the USA. Loss of blood supply to the brain due to arterial blockage results in a cascade of damaging events such as excitotoxicity and oxidative stress that eventually re- sult in brain damage and neurological deficit [1–3]. The un- derlying gene-expression changes that occur during an ische- mic attack have been under intense focus over the last few decades [4–12]. A better understanding of the brain’ s innate molecular response to ischemia will improve our understand- ing of the pathophysiology of stroke and help identify novel targets for effective neuroprotective strategies against the stroke injury. To date, nearly all of the high-throughput studies evaluating transcriptomic profiles in experimental stroke have utilized probe-based techniques such as microarray [4, 5, 7, 12–15]. Although microarrays have contributed significantly to our understanding of gene expression changes in the post- stroke brain, this methodology is limited in that one can only explore the changes in the expression of previously known transcripts for which probes have been incorporated onto the microarray. This precludes the assessment of a vast portion of the unannotated transcriptome. Recent advances in genome- wide sequencing have enabled us to overcome this limitation and probe deeper into the transcriptome. Taking advantage of this technological advance, we recently published the first study on the genome-wide evaluation of the noncoding tran- scriptome in the post-stroke cerebral cortex in which we Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12035-018-1147-0) contains supplementary material, which is available to authorized users. * Ashutosh Dharap ashutosh.dharap@hackensackmeridian.org 1 Laboratory for Stroke Research and Noncoding RNA Biology, JFK Neuroscience Institute, HackensackMeridian Health JFK Medical Center, 65 James Street, Edison, NJ 08820, USA Molecular Neurobiology https://doi.org/10.1007/s12035-018-1147-0