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Constitutively active Gli2 co-IP/MS in ciliated and unciliated cells

ABSTRACT: Co-IP/MS of HA-Gli2 (P1-6A), the constitutively active mutant of Gli2 in NIH/3T3 Flp-in cells. The cells were either ciliated or depleted of cilia by expression of the dominant negative mutant of Kif3a (dnKif3a). Sample 8033:A is ciliated cells, sample 8033:B is unciliated cells.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Pawel Niewiadomski

PROVIDER: MSV000093739 | MassIVE | Sat Dec 30 04:59:00 GMT 2023

REPOSITORIES: MassIVE

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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 compare NGS-derived constitutively active RhoC mutant (Q63E) overexpressed bladder cancer cells (T24) transcriptome profiling (RNA-seq) to quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Methods: Bladder cancer cells mRNA profiles of vector(VEC) and RhoC constitutively active mutant (Q63E) stably expressing T24 cells were generated by deep sequencing, in triplicate, using Illumina HiSeqX Ten. The sequence reads that passed quality filters were analyzed at the transcript isoform level with the method: Hisat2 2.1.0 followed by StringTie. qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: Using an optimized data analysis workflow, we mapped about 50 million sequence reads per sample to the hunam reference genome and identified 209,506 transcripts in the vector(VEC) and constitutively active RhoC mutant (Q63E) transduced bladder cancer cells (T24 cells) with Hisat2 2.1.0 workflow. RNA-seq data confirmed stable expression of known housekeeping genes. Approximately 3% of the transcripts showed differential expression between the vector(VEC) and constitutively active RhoC mutant (Q63E) transduced bladder cancer cells (T24 cells), with a fold change ≥1.5 and p value <0.05. Altered expression of 13 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 RhoC function in bladder cancer. Data analysis with Hisat2 2.1.0 workflow revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions: Our study represents the first detailed analysis of constitutively active RhoC mutant (Q63E) overexpressed bladder cancer cells 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 line. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

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Constitutively active Gli2 co-IP/MS in ciliated and unciliated cells

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