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| Status |
Public on Feb 08, 2023 |
| Title |
Next Generation Sequencing Facilitates Quantitative Analysis of TRAP from grafted human neural stem cells in stroke and naïve rat brains |
| Organisms |
Homo sapiens; Rattus norvegicus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to access grafted human neural stem cells and host tissue transcriptomes
Methods: Grafted mRNA profiles of 7 days transplanted hNSC were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500. The sequence reads that passed quality filters were analyzed at the transcript isoform level with Burrows–Wheeler Aligner (BWA) followed by RNA-Seq by Expectation Maximization (RSEM). qRT–PCR validation was performed using TaqMan and SYBR Green assays
Results: Using an optimized data analysis workflow, we mapped: 1) about 230 million sequence reads per sample to the human genome (build hg19) and identified 17,463 transcripts in the grafted hNSC; and 2) about 30 million sequence reads per sample to the rat genome (build rn5) and identified 13,4688 transcripts in the host tissues; using RSEM workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 4 housekeeping genes were validated with qPCR. RNA-seq data had a linear relationship with qPCR for more than four orders of magnitude. Approximately 14% of the transcripts showed differential expression between the grafted hNSC in naive and stroke; and approximately 41% of the transcripts showed differential expression between the naive and stroke tissues, with p value <0.05. Altered expression of over 10 genes was confirmed with qPCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to grafted hNSC and host function. Data analysis with TRAP and RSEM workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling.
Conclusions: Our study represents the first detailed analysis of grafted hNSC transcriptome, with biologic replicates, generated by TRAPseq technology. TRAPseq and RSEM could be applied to many xenograft cell transplantation paradigms. With this approach we can start to predict upstream regulators that signal between the host and graft, and to predict the downstream signaling pathways and biological processes these upstream regulators might affect.
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| Overall design |
TRAPseq of grafted hNSC after 7 days of transplantation into naïve and stroke nude rats
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| Web link |
https://www.cell.com/cell-reports/fulltext/S2211-1247(23)00364-9?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS2211124723003649%3Fshowall%3Dtrue
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| Contributor(s) |
Azevedo-Pereira RL, Bliss TM, Steinberg GK |
| Citation(s) |
37043353 |
| Submission date |
May 17, 2020 |
| Last update date |
May 10, 2023 |
| Contact name |
Ricardo Luiz De Azevedo Pereira |
| E-mail(s) |
ricardo.azevedo@sanbio.com, ricoluiz@gmail.com
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| Organization name |
Stanford
|
| Department |
Neurosurgery
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| Lab |
Steinberg
|
| Street address |
1201 Welch Road
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platforms (2) |
| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
| GPL18694 |
Illumina HiSeq 2500 (Rattus norvegicus) |
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| Samples (20)
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| Relations |
| BioProject |
PRJNA633342 |
| SRA |
SRP262030 |
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