Project description:To analyse gene expression differences between HeLa tranfected cells with mutant COL1 (COL1G610C) compared to their relative scramble controls
Project description:Single cell RNA sequencing (scRNA-seq) has emerged as a versatile tool in biology, enabling comprehensive genomic-level characterization of individual cells. Currently, most scRNA-seq methods generate barcoded cDNAs by capturing polyA tails of mRNAs, which excludes many non-coding RNAs (ncRNAs), especially those transcribed by RNA polymerase III (Pol III). Although previously thought to be expressed constitutively, Pol III-transcribed ncRNAs are expressed variably in healthy and disease states and play important roles therein, necessitating their profiling at the single cell level. In this study, we have developed a measurement protocol for nc886 as a model case, as an initial step for scRNA-seq for Pol III-transcribed ncRNAs. Specifically, we spiked in an oligo-tagged nc886-specific primer during the polyA tail capture process for the 5’-reading in scRNA-seq. We then produced sequencing libraries for standard 5’ gene expression and oligo-tagged nc886 separately, to accommodate different cDNA sizes and ensure undisturbed transcriptome analysis. We applied this protocol in three cell lines which express high, low, and zero levels of nc886, respectively. Our results show that the identification of oligo tags exhibited limited target specificity, and sequencing reads of nc886 enabled the correction of non-specific priming. These findings suggest that gene-specific primers (GSPs) can be employed to capture RNAs lacking a polyA tail, with subsequent sequence verification ensuring accurate gene expression counting. Moreover, we embarked on an analysis of differentially expressed genes in cell line sub-clusters with differential nc886 expression, demonstrating variations in gene expression phenotypes. Collectively, the primer spike-in strategy allows us for a combined analysis of ncRNAs and gene expression phenotype.
Project description:Transforming growth factor-β (TGF-β) signaling and microRNAs (miRNAs) are important gene regulatory components in cancer. Usually in advanced malignant stages, TGF-β signaling is elevated, but global miRNA expression is suppressed. Such a gene expression signature is well illustrated in a fibrosis/mesenchymal subtype of ovarian cancer (OC) that is of poor prognosis. However, the interplay between the two pathways in the OC subtype has not yet been elucidated. nc886 is a recently identified non-coding RNA implicated in several malignancies. nc886's high expression is associated with the poor prognosis of 285 patients in an OC cohort. Herein we have found in OC that nc886 expression is induced by TGF-β and that nc886 binds to the enzyme Dicer to inhibit the processing of miRNA precursors into mature forms. By preventing the miRNA pathway, nc886 emulates TGF-β in gene expression patterns and potentiates cell adhesion, migration, invasion, and drug resistance. We report nc886 as a novel molecular link between the TGF-β and miRNA pathways.
Project description:To analyse differential expression genes（LncRNA and mRNA） in ATG7 knockdown cells and control cells upon influenza virus infection, transcriptome RNA sequencing was performed
Project description:nc886 is a 101 nucleotides long non-coding RNA that is also known as a precursor microRNA or a vault RNA. nc886 has been suggested to be a tumor suppressor, mainly inferred by its expression pattern as well as its genomic location at human chromosome 5q31, a locus for a tumor suppressor gene(s). In order to understand potential roles of nc886 in gastric cancer, we performed the knockdown of nc886 in nonmalignant HFE-145 cells and identified and analyzed genes whose expression is activated by nc886 knockdown in gastric cancer.
Project description:object:compare the genes influenced by TTC22 overexpression or knockdown Methods: For whole-genome transcriptome profiling, four libraries were generated from total RNA samples extracted from Flag-TTC22-OE, empty vector control, scramble RNA control, and TTC22-KD (siTTC#1&2&3) HCT116 cells using TruSeq® RNA Sample Preparation Kit (Illumina Inc.) according to the manufacturer’s protocol, and sequenced on the Illumina HiSeq PE150 platform (Genminix, Shanghai, China) using the 150–base pair single-end sequencing module. Hisat2 (version:2.0.4) was used to map the cleaned reads to the human hg38 reference genome. Results: The mRNA data obtained from transcriptome sequencing, subjected to T-test statistics and corrected by the RVM model. Significant differential mRNA (TwoClassDif) between TTC22-OE and vector control cells or between TTC22-KD and scramble RNA control cells was yielded. Up-regulated and down-regulated genes (fold change>1.5, calculated through Ballgown algorithm) were performed to find significant functions and significant signaling pathways based on the Gene Ontology database (GO-Analysis) or KEGG database.(Pathway-Analysis). Conclusion:The results suggested that TTC22 could affect the RNA metabolism pathway that controls the progression and metastasis of CC.
