Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Next generation sequencing of the transcriptome in MCF-7 cells with/without SRA knockdown

ABSTRACT: We employed next generation sequencing to examine whether knocking down the steroid receptor RNA activator (SRA) gene significantly affect the expression levels of certain genes in MCF-7 cells. MCF-7 cells were transfected with either a pool of four non-target control siRNAs or a pool of four SRA siRNAs for 32 hrs. 157 million reads were generated from triplicate samples of the control group; 151 million reads were generated from triplicate samples of the SRA knockdown group. Six genes were identified as significantly changed in the expression levels with the cutoff of q value ≤ 0.05, fold change ≤ 0.5 or ≥ 2, and reads per kilobase per million mapped reads (RPKM) ≥ 1. However, except for SRA itself, the other five genes were shown by real-time PCR to be only affected by one siRNA in the SRA siRNA pool. Further analysis of this dataset with different cuttoff setting may reveal true SRA-regulated genes in MCF-7. MCF-7 cells were cultured in high glucose DMEM with 10% fetal bovine serum, 2 mM Glutamax-1, 100 units/ml penicillin and 100 μg/ml streptomycin. ON-TARGETplus SMARTpool for human SRA (Thermo Scientific, L-027192-00-0005) was used to knockdown SRA (siSRA) and ON-TARGETplus Non-targeting Pool Thermo Scientific, D-001810-10-05) was used as a negative control (siCtrl). A total of 25 nM siRNA was transfected in 6-well dishes using Lipofectamine™ RNAiMAX Reagent (Life Technologies, Invitrogen) following the manufacturer’s recommendations. Polyadenylated RNA was purified from the cells 32 hrs after transfection. cDNA libraries were prepared and double-stranded cDNA was fragmented using DNase I according to Illumina specifications, prior to adaptor ligation. Sequencing libraries were amplified and sequenced using an Illumina HiSeq 2000 sequencer.

ORGANISM(S): Homo sapiens

SUBMITTER: Linghe Xi

PROVIDER: E-GEOD-52385 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

Structure and function of steroid receptor RNA activator protein, the proposed partner of SRA noncoding RNA.

McKay David B DB Xi Linghe L Barthel Kristen K B KKB Cech Thomas R TR

Journal of molecular biology 20140130 8

In a widely accepted model, the steroid receptor RNA activator protein (SRA protein; SRAP) modulates the transcriptional regulatory activity of SRA RNA by binding a specific stem-loop of SRA. We first confirmed that SRAP is present in the nucleus as well as the cytoplasm of MCF-7 breast cancer cells, where it is expressed at the level of about 10(5) molecules per cell. However, our SRAP-RNA binding experiments, both in vitro with recombinant protein and in cultured cells with plasmid-expressed p ...[more]

PMID: 24486609

Similar Datasets

Project description:Background: MYC is a transcription factor encoded by the c-MYC gene (thereafter termed MYC). MYC is key transcription factor involved in many central cellular processes including ribosomal biogenesis. MYC is overexpressed in the majority of human tumours including aggressive B-cell lymphoma especially Burkitt's lymphoma. Although Burkitt's lymphoma is a highlight example for MYC overexpression due to a chromosomal translocation, no global analysis of MYC binding sites by chromatin immunoprecipitation (ChIP) followed by global next generation sequencing (ChIP-Seq) has been conducted so far in Burkitt's lymphoma. Methodology/Principal Findings: ChIP-Seq was performed with a MYC-specific antibody giving rise to 7,054 predicted MYC binding sites after bioinformatics analysis of a total of 19 million sequence reads. In line with previous findings, binding sites accumulate in gene sets known to be involved in the ribosomal biogenesis, histone acetyltransferase and methyltransferase complexes and the cell cycle demonstrating a regulatory role of MYC in these processes. Unexpectedly, MYC binding sites also accumulate in genes typically expressed in mature B-cells. To assess the functional consequences of altered MYC binding, the ChIP-Seq data were supplemented with siRNA mediated knock-downs of MYC in BL cell lines followed by gene expression profiling. Interestingly, amongst others, genes involved in B-cell function were up-regulated in response to MYC silencing. Conclusion/Significance: The 7,054 MYC-binding sites identified by our ChIP-Seq approach greatly extend the knowledge regarding MYC binding in Burkitt's lymphoma and sheds further light on the enormous complexity of the MYC regulatory network. Especially our observation that (i) many B-cell relevant genes are targeted by MYC and (ii) that MYC down-regulation leads to an up-regulation of B-cell genes highlights an interesting aspect of BurkittM-BM-4s lymphoma biology. [ChIP-Seq] Analysis of MYC DNA binding sites by ChiP-Seq in 5 BurkittM-BM-4s lymphoma cell lines (Raji, Ramos, Blue1, BL41, CA46) [mRNA expression profiling] siRNA-mediated knock-down of MYC was done employing the BL cell lines Raji, BL41 and Blue1 in order to detect MYC-driven gene expression changes. For this purpose, the cells were Amaxa-transfected using MYC smart pool siRNA and control siRNA (Thermo Scientific/Dharmacon, Erembodegem, Belgium), respectively.

Project description:Purpose: Zinc Finger MIZ-Type Containing 1 (Zmiz1) is a member of the PIAS family of protein and function as a transcriptional coactivator of Notch, Androgen Receptor (AR), p53, Estrogen Receptor (ER), and Smad3/4 . Despite Zmiz1 critical role in angiogenesis, its role in lymphatic vasculature is unknown. Here, we use HDLECs cell line to profile transcriptional changes upon Zmiz1 knockdown using siRNA. Methods: Total RNA was extracted from control and Zmiz1 siRNA transfected HDLECs using GeneJET RNA Purification Kit (Thermo Fisher Scientific, K0732) following the manufacturer instructions. RNA concentration and RNA integrity (RIN) number were determined using Qubit RNA High Sensitivity Assay Kit (Thermo Fisher Scientific, Q32852) and Bioanalyzer RNA 6000 Nano assay kit (Agilent, 5067-1511) respectively. RNA library was prepared using TruSeq RNA Library Prep Kit v2 (Illumina, RS-122-2001) according to the manufacturer instructions. mRNA library was quantified using Qubit dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific, Q32851) and verified using the Bioanalyzer DNA1000 assay kit (Agilent, 5067-1505). Verified samples were sequenced using the NextSeq1000/2000 P2 Reagents (200 Cycles) v3 (Illumina, 20046812) on a Nextseq1000/2000. Sequenced reads were aligned to the human (hg19) reference genome with RNA-Seq alignment tool (STAR aligner). The aligned reads were used to quantify mRNA expression and determine differentially expressed genes using the RNA-Seq Differential Expression tool (version 1.0.1). Both alignment and differential expression analysis were performed using the tools in the illumina BaseSpace Sequence Hub. Results: We found 2,228 differentially expressed genes of which 1,115 genes were upregulated while 1,113 genes were downregulated. Downregulated genes were enriched in biological processes such as lymph vessel development, cell migration and heart valve development. Conclusions: We identify Zmiz1 regulates Prox1 in lymphatic endothelial cells

Dataset Information

Next generation sequencing of the transcriptome in MCF-7 cells with/without SRA knockdown

Publications

Structure and function of steroid receptor RNA activator protein, the proposed partner of SRA noncoding RNA.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets