Phospho-RNA sequencing with CircAID-p-seq
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA731473 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| Other | ||||
| SRR14602119_1.fastq.gz | Fastqsanger.gz | |||
| SRR14602120_1.fastq.gz | Fastqsanger.gz | |||
| SRR14602122.fastq.gz | Fastqsanger.gz | |||
| SRR14602123_1.fastq.gz | Fastqsanger.gz |
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Project description:Accurate positional information concerning ribosomes and RNA binding proteins with respect to their transcripts is important to understand the global regulatory network underlying protein and RNA fate in living cells. Most footprinting approaches generate RNA fragments bearing a phosphate or cyclic phosphate groups at their 3′ end. Unfortunately, all current protocols for library preparation rely only on the presence of a 3′ hydroxyl group. Here, we developed circAID-p-seq, a PCR-free library preparation for 3′ phospho-RNA sequencing. We applied circAID-p-seq to ribosome profiling, which produces fragments protected by ribosomes after endonuclease digestion. CircAID-p-seq, combined with the dedicated computational pipeline circAidMe, facilitates accurate, fast, highly efficient and low-cost sequencing of phospho-RNA fragments from eukaryotic cells and tissues. While assessing circAID-p-seq to portray ribosomes engaged with transcripts, we provide a versatile tool to unravel any 3′-phospho RNA molecules.
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Project description:Most RNA footprinting approaches that require ribonuclease cleavage generate RNA fragments bearing a phosphate or cyclic phosphate group at their 3' end. Unfortunately, current library preparation protocols rely only on a 3' hydroxyl group for adaptor ligation or poly-A tailing. Here, we developed circAID-p-seq, a PCR-free library preparation for selective 3' phospho-RNA sequencing. As a proof of concept, we applied circAID-p-seq to ribosome profiling, which is based on sequencing of RNA fragments protected by ribosomes after endonuclease digestion. CircAID-p-seq, combined with the dedicated computational pipeline circAidMe, facilitates accurate, fast and highly efficient sequencing of phospho-RNA fragments from eukaryotic cells and tissues. We used circAID-p-seq to portray ribosome occupancy in transcripts, providing a versatile and PCR-free strategy to possibly unravel any endogenous 3'-phospho RNA molecules.
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Project description:Purpose: The goal of this study was to generate mRNA profiles for MCF7 breast cancer cells expressing phospho-deficient STAT5a point mutants with treatment of prolactin (PRL) to determine differential global genomic effects in breast cancer. This data was compared to in vitro studies using the same cells to determine phenotypic outcomes of the STAT5a point mutations. Methods: mRNA profiles of prolactin (PRL) treated or untreated MCF7 cells expressing wild type (WT)-STAT5a, or phospho-deficient point mutants Y694F-, S726A-, or S780A-STAT5a were generated in triplicate by sequencing on the S4 flow cell platform of the Illumina NovaSEQ 6000 System (Illumina, Inc), generating ≥50 million 150 bp paired-end (PE) reads per sample. All libraries were prepared and sequenced in a single batch/flow cell lane. Results: RNA-sequencing and subsequent Ingenuity Pathway Analysis predicted that loss of each phospho-site differentially affected both prolactin-induced gene expression as well as functional pathways of breast cancer (e.g. cell survival, proliferation, and colony formation). Gene set analysis for transcription factor activity (using ENCODE project ChIP-sequencing and the web platform Enrichr), illustrated multiple transcription factors at work in MCF7 cells expressing each phospho-deficient STAT5a point mutant, indicating that these point mutants have roles regulating STAT5a activity at various enhancer/promoters in a enhancer/promoter specific context. Conclusion:
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Project description:RNA-seq analysis of mRNAs immunoprecipitated by wild-type (WT), phospho-mimetic (S200D), or phospho-null (S200A) FLAG-LIN28A
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Project description:Discovering noncanonical peptides has been a common application of proteogenomics. Recent studies suggest that certain noncanonical peptides, known as ncMAPs (noncanonical MHC-I-associated peptides), that bind to major histocompatibility complex I may make good immunotherapeutic targets. De novo peptide sequencing is a great way to find ncMAPs since it can detect peptide sequences from their tandem mass spectra without using any sequence databases. However, this strategy hasn’t been widely applied for ncMAP identification because there is not a good way to estimate its false-positive rates. In order to completely and accurately identify immunopeptides using de novo peptide sequencing, we describe a unique pipeline called pXg. In contrast to current pipelines, it makes use of genomic data, RNA-Seq abundance and sequencing quality, in addition to proteomic features to increase the sensitivity and specificity of peptide identification. We show that the peptide-spectrum match quality and genetic traits have a clear relationship, showing that they can be utilized to evaluate peptide-spectrum matches. From ten samples, we found 24,449 cMAPs (canonical MHC-I-associated peptides) and 956 ncMAPs by using a target-decoy competition. 387 ncMAPs and 1,611 cMAPs were novel identifications that had not yet been published. We discovered 11 ncMAPs produced from a squirrel monkey retrovirus in human cell lines in addition to the 2 ncMAPs originating from a complementarity determining region 3 in an antibody thanks to the unrestricted search space assumed by de novo sequencing. These entirely new identifications show that pXg can make the most of de novo peptide sequencing's advantages and its potential use in the search for new immunotherapeutic targets.
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Project description:Extracellular RNAs (exRNAs) in blood and other biofluids have attracted great interest as potential biomarkers in liquid biopsy applications, as well as for their potential biological functions. Whereas it is well-established that extracellular microRNAs are present in human blood circulation, the degree to which messenger RNAs (mRNA) and long noncoding RNAs (lncRNA) are represented in plasma is less clear. Here we report that mRNA and lncRNA species are present as small fragments in plasma that are not detected by standard small RNA-seq methods, because they lack 5’-phosphorylation or carry 3’-phosphorylation. We developed a modified sequencing protocol (termed “phospho-sRNA-seq”) that incorporates upfront RNA treatment with T4 polynucleotide kinase (which also has 3’ phosphatase activity) and compared it to a standard small RNA-seq protocol, using as input both a pool of synthetic RNAs with diverse 5’ and 3’ end chemistries, as well exRNA isolated from human blood plasma. This series uses the synthetic pools of small RNAs to demonstrate the efficacy of phospho-sRNA-seq to enable capture of sRNAs lacking a 5’ phosphate and/or having a 3’ phosphate.
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