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Next Generation Sequencing Facilitates Quantitative Analysis of VSV-stimulated Wild Type and lncRNA-ISIR -/- spleen Transcriptomes

ABSTRACT: Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. Methods: spleen mRNA profiles of 8-weeks-old WT and lncRNA-ISIR, used to be called lnRNA-IRF3, knockout mice were generated by deep sequencing, using BGISEQ-500 system. The sequence reads that passed quality filters were analyzed at the transcriptional level with RSEM (RNA-seq by Expectation Maximization). Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

ORGANISM(S): Mus musculus

PROVIDER: GSE138700 | GEO | 2021/11/09

REPOSITORIES: GEO

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Project description: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.

Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived thyroid cancer cell line k1 transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Methods: mRNA profiles of shCtrl and shTBX3 cells were generated by deep sequencing, in duplicate, using BGISEQ-500. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods:Bowtie2 to map clean reads to reference gene and use HISAT to reference genome.Bowtie2 parameters forSE reads:-q--phred64--sensitive--dpad0--gbar and HISTAparameters forSE reads:-p8--phred64--sensitive-I1-X 1000. qRT–PCR validation was performed using SYBR Green assays Results: Using an optimized data analysis workflow, we mapped about 24 million sequence reads per sample to the human genome (build hg19) and identified 20511 transcripts in K1-shCtrl and K1-shTBX3 cells with RSEM workflow. Approximately 5% of the transcripts showed differential expression between K1-shCtrl and K1-shTBX3 cells, with a fold change ≥1.5 and p value <0.01. Altered expression of 16 candidate genes was confirmed with qRT–PCR, 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 thyroid cancer function. Conclusions: Our study represents the first detailed analysis of thyroid cancer cell line k1 transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

Project description:Goal: High-throughput sequencing-by-synthesis (Illumina) RNA sequencing technology was carried out with an aim to gain deeper understanding of immune host protective mechanisms. Here, RNA-seq was applied to understand the differential gene expression profile of mice spleen following immunization with Brucella abortus S19∆per mutant (perosamine synthetase gene mutant of Brucella abortus S19) in comparison to mock immunized mice spleen (PBS inoculated). Methods: RNA-seq data of 15th day post immunized mice spleen (with Brucella abortus S19∆per) and PBS control mice were generated by deep sequencing ( in duplicate) using IlluminaNextSeq 500 . The sequence reads that passed quality filters were analyzed for transcript abundance using RSEM package (RNA-Seq by Expectation Maximization) (Li and Dewey, 2011). Breifly, the RSEM package generated a reference sequence based on given mouse transcript annotations (Mus_musculus.GrCm38.83.chr.gtf.gz). The Bowtie allignmet tool available within the package was used to calculate expected counts (number of mapped reads) using quality trimmed reads and reference sequence. Finally, the expected counts estimated by RSEM were fed into different DE package tools, such as DESeq2 (Love et al., 2014), edgeR (Robinson et al., 2010) and EBSeq (Leng et al., 2013) in order to identify differentially expressed genes across spleen samples (B. abortus S19∆per versus (vs) PBS control. Functional annotation of differently expressed genes were carried out using g:Profiler (Reimandet al., 2016; http://biit.cs.ut.ee/gprofiler/). Results: A mean of 37.58 million processed reads (range: 30.51 million to 51.79 million reads per individual RNA-seq library) were generated during the experiment. The expected counts generated by the RSEM package followed by differential analysis calculation using different DE packages identified a total of 1917 differentially expressed genes (DEGs), of which 968 and 949 genes were up- and down-regulated respectively. Functional annotation revealed 545 significantly enriched genes to be associated with immune system processes within the total 1917 differentially expressed genes. Further analysis revealed 21 genes showing significant expression were also in MHC-I and MHC-II antigen processing and presentation pathway during S19∆per immunization. Conclusions:The RNA-seq data revealed the coordinated up-regulation of MHC-I and MHC-II processing pathways providing insights into the molecular mechanism of immune protection conferred by B. abortus S19∆per in mice at day 15 post immunization and might aid in the development of new attenuated vaccine strains with improved efficacy.

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Next Generation Sequencing Facilitates Quantitative Analysis of VSV-stimulated Wild Type and lncRNA-ISIR -/- spleen Transcriptomes

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