Project description:To evaluate the effect of mouse Siglec1 on B16F10 melanoma we used co-culture methos where HEK293T cells were transiently transfected with plasmid containg mouse Siglec1 or empty plasmid (mock). These HEK293T cells were cultured with B16F10-GFP mouse melanoma cells for 18 hours in low binding plates. After 18 hours of incubation on 37°C, 5% CO2, melanoma cells were FACS sorted based on GFP expression and total RNA was isolated and used for TruSeq RNA library construction for transcriptome analysis. Samples were from 3 independent experiments. The library preparation and NGS data aquisition were performed by Macrogen (Macrogen, Inc., Korea)
Project description:We investigated the transcriptome profile of HEK293T cells transfected with a plasmid encoding 9J10, a peptide isolated in a phenotypic screen from a library of peptides derived from bacterial and archaeal genomes. The peptide was identified in a screen for FOXO3a reactivation and has been shown to interact with 14-3-3 proteins and induce relocalisation of FOXO3a from the cytoplasm to the nucleus. A peptide with a single amino acid mutation does not interact with 14-3-3 and was included as a control. A constitutively active FOXO3a mutant was also included as a control.
Project description:ING1b and GADD45a are nuclear proteins involved in the regulation of cell growth, apoptosis and DNA repair. We found that ING1b and GADD45a physically and functionally interact in the epigenetic regulation of specific target genes. In order to characterise the functional ING1b-GADD45a interaction, we performed a gain-of-function experiment in HEK293T cells by individual and combinatorial plasmid transfections and then analysed the transcriptional response via expression microarray profiling. HEK293T cells were transiently transfected with expression plasmids encoding human GADD45a and/or human ING1b (full-length or without its PHD-domain) and harvested 48h post-transfection for Illumina microarray profiling. Two independently transfected replicate samples of each condition were analysed. Empty vector (control) transfections served as reference samples.
Project description:To determine what effect the collateral activity of RfxCas13d has on cells, a HEK293T cell line stably expressing RfxCas13d (HEK293T-RfxCas13d) was constructed and then transfected with plasmids encoding target gene and corresponding crRNAs. After 24 hours of transfection, cells were collected and extracted for total RNA. Then, we performed RNA-seq to compare the differentially expressed genes between cells transfected with targeting crRNAs and non-targeting crRNA.
Project description:Overexpression of USF1 in HEK293T cells in vitro to ascertain the genes downstream of USF1. Will identify direct targets as well as indirect targets of USF1. Keywords: Overexpression of transcription factor to determine downstream targets. Cloned USF1 from cDNA and transiently transfected into HEK293T cells. Total RNA collected 48 hours after transfection. Empty vector used as control. Expression of USF1 was validated using western blot, and qRT-PCR was used to validate differential expression.
Project description:Purpose: Comparison of RNA-sequencing datasets obtained from exosomes of Nef-transfected and Mock-transfected HEK293T cells Methods: Assessment of RNA content of exosomes produced by Nef-transfected HEK293T cells and and Mock-transfected HEK293T cells Results: Differences in a set of microRNAs Conclusions: Nef-transfection induces changes in the microRNA content of exosomes
Project description:Purpose: Comparison of RNA-sequencing datasets obtained from exosomes of Nef-transfected and Mock-transfected HEK293T cells. Methods: Assessment of RNA content of exosomes produced by Nef-transfected HEK293T cells and and Mock-transfected HEK293T cells. Results: Differences in a set of mRNAs. Conclusions: Nef-transfection might induces changes in the mRNA content of exosomes.
Project description:HEK293T cell expression data from four conditions: mock transfection, Ago2 transfection, Ago2 & miR-1 transfection, and Ago2 & miR-124 transfection. RNA was isolated directly from 10cm dishes using Trizol reagent and amplified. Set of arrays that are part of repeated experiments Compound Based Treatment: FLAG-Ago2 transfection Keywords: Biological Replicate
Project description:MicroRNAs (miRNAs) are a class of small RNA molecules that regulate expression of specific mRNA targets. They can be released from cells, often encapsulated within extracellular vesicles (EVs), and therefore have the potential to mediate intercellular communication. It has been suggested that certain miRNAs may be selectively exported, although the mechanism has yet to be identified. Manipulation of the miRNA content of EVs will be important for future therapeutic applications. We therefore wished to assess which endogenous miRNAs are enriched in EVs and how effectively an overexpressed miRNA would be exported. Small RNA libraries from HEK293T cells and vesicles before or after transfection with a vector for miR-146 overexpression were analysed by deep sequencing. A subset of miRNAs was found to be enriched in EVs. The global expression data provided by deep sequencing confirms that specific miRNAs are enriched in EVs released by HEK293T cells. Cells were transfected with a plasmid to direct overexpression of miR-146a. Extracellular vesicles were isolated by ultracentrifugation from untreated and transfected cells. RNA was isolated from one sample each of untreated and transfected cells and vesicles.Small RNA libraries were prepared for sequencing.
