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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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE52385 | GEO | 2014/01/23

SECONDARY ACCESSION(S): PRJNA227746

REPOSITORIES: GEO

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

Project description: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.

2014-01-23 | E-GEOD-52385 | biostudies-arrayexpress

Expression Profiling Reveals Unexpected Targets and Functions of the Human Steroid Receptor RNA Activator (SRA) Gene

Project description:The human steroid receptor RNA activator (SRA) gene encodes both non-coding RNAs (ncRNAs) and protein-generating isoforms. However, the breadth of endogenous target genes that might be regulated by SRA RNAs remains largely unknown. To address this, we depleted SRA RNA in two human cancer cell lines (HeLa and MCF-7) with small interfering RNAs, then assayed for changes in gene expression by microarray analyses using Affymetrix HGU133+2 arrays. We also tested if SRA depletion affects estradiol-regulated genes in MCF-7 breast cancer cells. We transiently transfected HeLa or MCF-7 human cancer cell lines with with non-targeting (NT) or SRA-targeting siRNAs. Six total HeLa RNA samples were analyzed on an Affymetrix HGU133+2 array, representing three biological triplicates. Likewise, 12 total MCF-7 RNA samples were analyzed on Affymetrix HGU133+2 arrays, representing biological trilplicates for both NT and siSRA transfected cells and with/without 10 nM estradiol (E2) for 6 hours.

2010-02-21 | E-GEOD-20081 | biostudies-arrayexpress

Expression Profiling Reveals Unexpected Targets and Functions of the Human Steroid Receptor RNA Activator (SRA) Gene

2010-02-01 | GSE20081 | GEO

SRA Example BioProject

Project description:SRA Example BioProject

| PRJNA248299 | ENA

RNAseq analysis of A549 cells depleted of RPS19 using siRNAs

Project description:Gene expression profiling analysis of RNA-seq data for A549 cells transfected with non-targeting siRNA (siOTP) and KD derivatives (siRPS19) to ascertain relative gene expression (reads per kilobase per million, RPKM) for use to intersect with high-throughput RNA interference screening (siRNA) data to assess baseline expression of candidates being screened in these assays.

2022-09-30 | GSE212775 | GEO

Genome wide RNA sequencing to profile response to IL-1 in control and IFT88 depleted cells

Project description:Purpose: RNA sequencing across whole genome downstream to IL-1 to investigate scope and nature of effect of IFT88 depletion Method: Control pool treated and IFT88 siRNA pool targeted Fibroblast-like chondrocytes were treated with or without IL-1B 10ng/ml and RNA collected in triplicate at 0, 1, 2, 4, 8 and 24h. PolyA-selected sequencing libraries were prepared using the TruSeq protocol (Illumina). Libraries were subject to 75bp paired-end sequencing (Illumina HiSeq 4000) to an average depth of 28.5 million read pairs per sample and aligned to mouse genome with Hisat2. Results: Using time-course analysis (ImpulseDE2) the IL-1 response timecourse was determined (control-only) and intersected against the comparative case vs control analysis. Per-point differential expression analysis IFT88 vs control was performed using DESeq2. Conclusion: Demonstration of the effect of IFT88 depletion on IL-1 response signature over 24h.

2020-07-02 | GSE128532 | GEO

RNA-seq analysis of control and NRDE2-depleted breast cancer cells

Project description:Purpose: investigate transcriptomic changes following depletion of the NRDE2 gene Methods: MDA-MB-231 breast cancer cells were transfected with 20nM control or NRDE2-targeting siRNAs, and RNA was collected after 48h for RNA-seq analysis Results: We analyzed si-Control and si-NRDE2-treated samples in triplicate (~46-64 million reads per sample) and find that the near-complete loss of NRDE2 results in generally modest changes in gene expression, with only 84 genes deregulated >2-fold. We also identify 342 introns which are differentially retained in NRDE2-depleted cells. Conclusions: NRDE2 is required for suppressing intron retention in a subset of genes

2018-09-30 | GSE119827 | GEO

Multiple Roles for Steroid Receptor RNA Activator (SRA) in Regulation of Adipogenesis and Insulin Sensitivity

Project description:PPARγ is a master transcriptional regulator of adipogenesis. Hence, the identification of PPARγ coactivators should help reveal mechanisms controlling gene expression in adipose tissue development and physiology. We show that the non-coding RNA Steroid receptor RNA Activator, SRA, associates with PPARγ and coactivates PPARγ-dependent reporter gene expression. Overexpression of SRA in ST2 adipocyte precursor cells promotes their differentiation into adipocytes. Conversely, knockdown of endogenous SRA inhibits 3T3-L1 preadipocyte differentiation. Microarray analysis reveals hundreds of SRA-responsive genes in adipocytes, including genes in cell cycle, insulin and TNFα signaling pathways. Some functions of SRA may involve mechanisms other than coactivation of PPARγ. SRA increases insulin-stimulated glucose uptake in adipocytes. SRA promotes S-phase entry during mitotic clonal expansion, decreases expression of cyclin-dependent kinase inhibiters p21Cip1 and p27Kip1, and increases phosphorylation of Cdk1/Cdc2. SRA also inhibits the TNFα-induced phosphorylation of c-Jun NH2-terminal kinase. In conclusion, SRA enhances adipogenesis and adipocyte function through multiple pathways.

2013-05-07 | GSE21594 | GEO

Pan-cancer transcriptomic analysis associates long non-coding RNAs with key mutational driver events

Project description:To identify genes regulated by NFE2L2 (Nrf2), we selected a lung cancer cell line (A549) in which NFE2L2 is normally active. Three transfections using siRNAs targeting NFE2L2 and four control transfections using two different negative control siRNAs were done. As a result, we found several genes up or down regulated in response to NFE2L2 inactivation in these cells. A549 cells were cultured, and 250,000 cells were transfected with Lipofectamine RNAiMAX reagent containing 5 nmol siRNA particles in a 6-well plate. Three transfections with siRNAs targeting NFE2L2 were performed, using three independent duplexes, while four control transfections were done using two different control duplexes, in addition to one mock (Lipofectamine only) transfection. RNA was prepared using the RNeasy Mini kit. 8 strand-specific total RNA-seq libraries were generated on an Illumina NextSeq 500. Between 50.5 and 60.6 million reads were obtained per sample. Reads were aligned to hg19 genome, at an alignment rate exceeding 94.4% for all samples.

2016-10-25 | E-GEOD-75452 | biostudies-arrayexpress

Multiple Roles for Steroid Receptor RNA Activator (SRA) in Regulation of Adipogenesis and Insulin Sensitivity

Project description:PPARM-NM-3 is a master transcriptional regulator of adipogenesis. Hence, the identification of PPARM-NM-3 coactivators should help reveal mechanisms controlling gene expression in adipose tissue development and physiology. We show that the non-coding RNA Steroid receptor RNA Activator, SRA, associates with PPARM-NM-3 and coactivates PPARM-NM-3-dependent reporter gene expression. Overexpression of SRA in ST2 adipocyte precursor cells promotes their differentiation into adipocytes. Conversely, knockdown of endogenous SRA inhibits 3T3-L1 preadipocyte differentiation. Microarray analysis reveals hundreds of SRA-responsive genes in adipocytes, including genes in cell cycle, insulin and TNFM-NM-1 signaling pathways. Some functions of SRA may involve mechanisms other than coactivation of PPARM-NM-3. SRA increases insulin-stimulated glucose uptake in adipocytes. SRA promotes S-phase entry during mitotic clonal expansion, decreases expression of cyclin-dependent kinase inhibiters p21Cip1 and p27Kip1, and increases phosphorylation of Cdk1/Cdc2. SRA also inhibits the TNFM-NM-1-induced phosphorylation of c-Jun NH2-terminal kinase. In conclusion, SRA enhances adipogenesis and adipocyte function through multiple pathways. Total RNA was isolated from fully differentiated (MDIT day 4) SRA overexpressing (pMSCV-SRA) and control (pMSCV empty vector) ST2 adipocytes, or fully differentiated (MDIT day 8) shSRA knockdown (pSuperior-shSRA) or shControl (pSuperior-shcontrol) 3T3-L1 adipocytes. Genome wide gene expression analysis was performed using Affymetrix mouse genome 430 2.0 arrays. Triplicate samples were analyzed.

2013-05-07 | E-GEOD-21594 | biostudies-arrayexpress

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