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Identifying LINC00899 binding sites in HeLa cells with CHART-seq

ABSTRACT: Identifying LINC00899 binding sites in HeLa cells with CHART-seq

PROVIDER: PRJEB29987 | ENA |

REPOSITORIES: ENA

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Identifying LINC00899 binding sites in HeLa cells with CHART-seq

Project description:LncRNAs represent a major transcriptional output of the human genome, but the function of many of these elements is unknown. For chromatin-localized lncRNAs, identification of genomic binding sites of these lncRNAs provides one opportunity to characterize their function. In this experiment, we have used capture hybridization analysis of RNA targets with high-throughput sequencing (CHART-seq) to identify the binding sites of the lncRNA LINC00899 in HeLa cells. LINC00899 is of interest as its depletion results in mitotic delay, suggesting a role in mitotic progression. This experiment contains 5 replicate batches where each batch contains a sample with antisense capture oligonucleotides (COs) to hybridize to and pull down the lncRNA transcript; an input control for the antisense pulldown; a control sample with sense COs, which should not hybridize to and pull down the lncRNA transcript; and an input control for the sense pulldown. All samples in the same batch were generated at the same time, and each pulldown (antisense or sense) was performed on chromatin obtained from independent cell cultures. All samples were subjected to high-throughput paired-end sequencing across two lanes.

2019-03-29 | E-MTAB-7418 | biostudies-arrayexpress

MYC binding sites in HeLa cells

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ENO1's RNA-binding sites in Human HeLa cells using eCLIP

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A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins (mRNA-seq)

Project description:Crosslinking and immunoprecipitation (CLIP) is increasingly used to map transcriptome-wide binding sites of RNA-binding proteins (RBPs). We developed a method for CLIP data analysis and applied it to compare 254 nm CLIP with PAR-CLIP, which involves crosslinking of photoreactive nucleotides with 365 nm UV light. We found small differences in the accuracy of these methods in identifying binding sites of HuR, a protein that binds low-complexity sequences and Argonaute 2, which has a complex binding specificity. We show that crosslink-induced mutations lead to single-nucleotide resolution for both PAR-CLIP and CLIP. Our results confirm the expectation from original CLIP publications that RNA-binding proteins do not protect sufficiently their sites under the denaturing conditions used during the CLIP procedure, and we show that extensive digestion with sequence-specific ribonucleases strongly biases the set of recovered binding sites. We finally show that this bias can be substantially reduced by milder nuclease digestion conditions. We performed duplicate mRNA-Seq experiments for cells grown in the presence of modified nucleotides or in normal medium, and following crosslinking at 365nm, 254nm or without crosslinking. In addition, we performed single mRNA-Seq experiments of cells transfected with anti-GFP or anti-HuR siRNA.

2011-05-15 | E-GEOD-28864 | biostudies-arrayexpress

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