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RNA-seq analysis of mRNAs immunoprecipitated by wild-type (WT) or phospho-null (S200A) FLAG-LIN28A in HeLa cells

ABSTRACT: To explore the effect of LIN28 (S200) phosphorylation on LIN28's association with mRNAs on a transcriptome-wide scale, we performed mRNA-seq on RNAs immunoprecipitated (RIP-seq) by wild-type (WT) or phospho-null (S200A) LIN28 in HeLa cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE83904 | GEO | 2016/10/28

SECONDARY ACCESSION(S): PRJNA327372

REPOSITORIES: GEO

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RNA-seq analysis of mRNAs immunoprecipitated by wild-type (WT) or phospho-mimetic (S200D) FLAG-LIN28A in PA1 cells

Project description:To explore the effect of LIN28 (S200) phosphorylation on LIN28's association with mRNAs on a transcriptome-wide scale, we performed mRNA-seq on RNAs immunoprecipitated (RIP-seq) by wild-type (WT) or phospho-mimetic (S200D) LIN28 in PA1 cells.

2016-10-28 | GSE83905 | GEO

RNA-seq analysis of mRNAs immunoprecipitated by wild-type (WT), phospho-mimetic (S200D), or phospho-null (S200A) FLAG-LIN28A

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RNA-seq analysis of mRNAs immunoprecipitated by wild-type (WT), phospho-mimetic (S200D), or phospho-null (S200A) FLAG-LIN28A

Project description:This SuperSeries is composed of the SubSeries listed below.

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LIN28A modulates splicing and gene expression programs in breast cancer cells [RIP-Seq]

Project description:The goals of this study were to identify LIN28 downstream gene targets in breast cancer cells. We use a subclone of the MCF-7 breast cancer cell line, MCF-7M as our model system. Methods: mRNA-protein complexes (mRNP) lysates were prepared from MCF-7M cells and incubated with Protein-A Sepharose beads (Sigma-Aldrich) and either LIN28 (Abcam) or control normal rabbit serum IgG antibodies. LIN28 interacting mRNAs were identified by whole genome sequencing. Results: Using an optimized data analysis workflow, we mapped approximately 13 million sequence reads for LIN28-IP and CTL- IP (IgG), respectively to the to the human genome (build h19). Conclusions: mRNA were significantly bound by LIN28 if LIN28 RIP had 2.5 fold increase in normalized reads compared to IgG. We found that LIN28 was predominantly bound at coding exons and 3'UTRs, 38% & 45% respectively, in the 843 mRNAs within MCF-7M genome.

2015-07-27 | GSE71009 | GEO

Sequencing of RNA fragments immunoprecipitated by anti-Flag in ALKBH5 overexpression NOMO1 cells

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Sequencing of RNA fragments immunoprecipitated by anti-Flag in CASC3 or MTDH overexpression breast cancer cells

Project description:RNA immunoprecipitated sequencings were conducted from CASC3 or MTDH overexpression breast cancer cells

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FLAG RIP-Seq identifies RNA species bound by cytoplasmic TAF7 in HeLa TAF7 (WT) cells

Project description:We perform FLAG RIP-Seq to generate a transcriptome-view of RNA targets bound by TAF7

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Embryonic neocortical HuR-immunoprecipitated mRNAs

Project description:Forkhead-box domain (Fox) containing family members are known autism spectrum associated genes. Here we show that, within the developing neocortex, the distinct phospho-states of a single RNA-binding protein, Hu antigen R, dictate mRNA translation and are sufficient to define distinct Foxp-characterized subpopulations of neocortical projection neurons. This demonstrates the importance of HuR phospho-states within the framework of the developing brain and further confirms the role of mRNA translation in autism pathogenesis.

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LIN28A modulates splicing and gene expression programs in breast cancer cells [RNA-Seq]

Project description:The goals of this study were to identify LIN28 downstream gene targets in breast cancer cells. We use a subclone of the MCF-7 breast cancer cell line, MCF-7M as our model system. Methods: mRNA profiles from MCF-7M breast cancer cells treated with siRNA against non-targeting control (NT), LIN28, hnRNP A1, LIN28 and hnRNPA1 (LIN28A1) for 72 hrs were generated by deep sequencing, in duplicate, using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: Using an optimized data analysis workflow, we mapped over 200 million sequence reads per sample to the human genome (build h19). Each of the four groups had two biological replicates. We developed a custom method to identify alternative splicing events and identified 111 genes with significant (FDR<0.05) differential splicing for LIN28 depleted cells compared to non-targeting siRNA control, as well as 249 and 182 genes for hnRNP A1 and LIN28A1 respectively. RNA-seq data were validated with by qRT–PCR analysis of a subset of genes. Conclusions: Results reveal that LIN28 regulates alternative splicing and steady state mRNA expression of genes implicated in aspects of breast cancer biology. Notably, cells lacking LIN28 undergo significant isoform switching of the ENAH gene, resulting in a decrease in the expression of ENAH exon 11a isoform. Expression of ENAH isoform 11a has been shown to be elevated in breast cancers that express HER2. mRNA profiles of MCF-7M cells treated with siRNA for NT control, LIN28, hnRNP A1, and LIN28 plus hnRNP A1 (A1) (LIN28A1) were generated by deep sequencing, in duplicate, using Illumina HiSeq 2000

2015-07-27 | E-GEOD-71012 | biostudies-arrayexpress

Genome-wide analysis of RNAs associated with Lin28

Project description:We used immunoprecipitation to pulldown Lin28 associated RNA targets and used genome-wide high throughput deep sequencing to identified those Lin28-associated RNAs. Keywords: Lin28 IP-RNAseq Examination of Lin28 immunoprecipitated RNA transcripts

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