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A unified strategy to clone modified/unmodified small RNAs and mRNAs for high throughput sequencing

ABSTRACT: A unified strategy to clone modified/unmodified small RNAs and mRNAs for high throughput sequencing

PROVIDER: PRJNA532412 | ENA |

REPOSITORIES: ENA

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A unified strategy to clone modified/unmodified small RNAs and mRNAs for high throughput sequencing

Project description:The high-throughput sequencing has become a standard tool for analyzing gene expression. Usually a cDNA/DNA library is constructed with 5' and 3' linkers and then sequenced. Unlike mRNA, small RNA often contains modifications including 5' cap or triphosphate and 3' methylation, affecting the linker addition during cloning processes. Small RNA is expressed at much lower levels than mRNA, making it more difficult to clone small RNA using a small amount of total RNA. Here we have developed a new strategy to clone modified/unmodified small RNA in an all-liquid-based reaction in a single PCR tube using as little as 20 ng total RNA. The 7-hour cloning process only needs ~1 hour labor time. Moreover, this method can be used to clone mRNA, simplifying the need to prepare two cloning systems for small RNA and mRNA. Since the linkers used are derived from the Illumina Truseq linkers for DNA cloning, the PCR primers based on the linker sequences can be used to obtain amplicons derived from small RNA/mRNA/DNA. Not only is our method more convenient for cloning modified RNA than available methods, but it is also more sensitive, accurate and versatile. Moreover, the all-liquid-based reaction can be performed in an automated manner.

2020-01-24 | GSE129664 | GEO

A unified model for the function of YTHDF proteins in the regulation of m6A-modified mRNA

Project description:N6-methyladenosine (m6A) is the most abundant mRNA nucleotide modification and regulates critical aspects of cellular physiology and differentiation. m6A is thought to mediate its effects through a complex network of interactions between different m6A sites and three functionally distinct cytoplasmic YTHDF m6A-binding proteins (DF1, DF2, and DF3). In contrast to the prevailing model, we show that DF proteins bind the same m6A-modified mRNAs, rather than different mRNAs. Furthermore, we find that DF proteins do not induce translation in HeLa cells. Instead, the DF paralogs act redundantly to mediate mRNA degradation and cellular differentiation. The ability of DF proteins to regulate stability and differentiation becomes evident only when all three DF paralogs are simultaneously depleted. Our studies reveal a unified model of m6A function in which all m6A-modified mRNAs are subjected to the combined action of the YTHDF proteins in proportion to the number of m6A sites.

2020-06-02 | GSE134380 | GEO

Mutational analysis of IVT mRNAs

Project description:RNA-seq analysis of in vitro transcribed mRNAs, containing modified nucleotides, or unmodified/canonical nucleotides, in order to assess the effect on the frequency of INDELS during transcription.

2023-10-13 | GSE223044 | GEO

A unified model for the function of YTHDF proteins in the regulation of m6A-modified mRNA

Project description:A unified model for the function of YTHDF proteins in the regulation of m6A-modified mRNA

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Comparative Ribo-Seq of Staphylococcus aureus harboring A2058-unmodified ribosomes and m6A2058-modified ribosomes

Project description:A total of 10 cDNA libraries were constructed, representing 3 and 2 independent biological replicates of S. aureus strain KES34 (unmodified ribosomes) and KES30 (m6A2058-modified ribosomes), respectively. Translational efficiency (RPF/mRNA ratios), total mRNA abundance and ribosome density of each ORFs were calculated.

2024-01-16 | GSE168265 | GEO

PTBP1 - Modified iCLIP

Project description:PTBP1 - Modified iCLIP

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eIF4A3 - Modified iCLIP

Project description:eIF4A3 - Modified iCLIP

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A unified single cell atlas of the mouse retina

Project description:Single cell RNA sequencing (scRNA-seq) has advanced the assessment of cellular heterogeneity at the single-cell resolution by identifying transcriptional similarities and differences. Data resources of scRNA-seq have been largely produced and extensively studied for the mouse retina. They serve as a powerful tool to study cellular components, transcriptome relationships, and regulatory mechanisms underlying various retinal diseases and biological processes. The large volume of mouse retinal scRNA-seq data has been released in separate repositories, limiting their widespread use in mouse retina communities. In this work, we are presenting a unified single-cell atlas for adult wild-type mouse retina using our in-house generated single-cell RNA-seq data complementing public datasets. The collected data account for over 323,000 single cells. After data integration, cell clustering, and cell type annotation, we have annotated 11 major classes and over 120 retinal cell types to form a unified single-cell reference for the mouse retina. To facilitate the public use of the reference, we have deposited it on CELLxGENE, UCSC Cell Browser, and Single Cell Portal for visualization and gene expression exploration. The unified atlas is also released to annotate new mouse retinal cells using scArches utilities. This unified reference serves an easy-to-use data resource of mouse retina communities.

2024-02-15 | GSE243413 | GEO

Identification of Mettl3-dependent m6A-modified mRNAs upon DNA-damage

Project description:The goal of the study was to identify those mRNAs that are m6A-modified in a Mettl3-dependent manner under DNA-damage condition in oncogene-expressing mouse embryonic fibroblasts

2022-07-07 | GSE169343 | GEO

Global m6A-modified mRNAs in the uterus of wild-type mouse

Project description:Global m6A-modified mRNAs in the uterus of wild-type mouse were analyzed by using methylated RNA immunoprecipitation sequencing (MeRIP-seq)

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