CRISPR/Cas9-targeted removal of unwanted sequences from small-RNA sequencing libraries
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE116029 | GEO | 2018/06/21
REPOSITORIES: GEO
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Project description:Single-cell transcriptomics (scRNA-seq) has revolutionized our understanding of cell types and states in various contexts, such as development and disease. Most methodology relies on poly(A) enrichment to selectively capture protein-coding polyadenylated transcripts, intending to exclude ribosomal transcripts that constitute >80% of the transcriptome. However, it is common for ribosomal transcripts to sneak into the library, which can add significant background by flooding libraries with irrelevant sequences. The challenge of amplifying all RNA transcripts from a single cell has motivated the development of new technologies to optimize retrieval of transcripts of interest. This problem is notable in planarians, where we find that 16S ribosomal transcripts were widely enriched (20-80%) across single-cell methods. Therefore, we adapted the Depletion of Abundant Sequences by Hybridization (DASH) to the standard 10X scRNA-seq protocol. We designed single-guide RNAs tiling the 16S sequence for CRISPR-mediated degradation, and subsequently generated untreated and DASH-treated datasets from the same libraries to enable a side-by-side comparison of the effects of DASH. DASH specifically removes 16S sequences without off-target removal of other genes. By assessing the cell barcodes shared by both libraries, DASH-treated cells have consistently higher complexity given the same amount of reads, which allows the detection of a rare cell cluster and more differentially expressed genes. In conclusion, DASH can be easily integrated into existing sequencing protocols and customized to deplete any unwanted transcripts.
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Project description:A major challenge for RNA-seq analysis of gene expression is to achieve sufficient coverage of informative non-ribosomal transcripts. In eukaryotic samples, this is typically achieved by selective oligo(dT)-priming of messenger RNAs to exclude ribosomal RNA (rRNA) during cDNA synthesis. However, this strategy is not compatible with prokaryotes in which functional transcripts are generally not polyadenylated. To overcome this, we adopted DASH (Depletion of Abundant Sequences by Hybridization), initially developed for eukaryotic cells, to improve both the sensitivity and depth of bacterial RNA-seq. DASH employs the Cas9 nuclease to remove unwanted cDNA sequences prior to library amplification. We report the design, evaluation, and optimization of DASH experiments for standard bacterial short-read sequencing approaches, including software for automated guide RNA (gRNA) design for Cas9-mediated cleavage in bacterial rDNA sequences. Using these gRNA pools, we effectively removed rRNA reads (56-86%) in RNA-seq libraries from two different model bacteria, the Gram-negative pathogen Salmonella enterica and the anaerobic gut commensal Bacteroides thetaiotaomicron. DASH works robustly, even with sub-nanogram amounts of input cDNA. Its efficiency, high sensitivity, ease of implementation, and low cost (~$5 per sample) render DASH an attractive alternative to rRNA removal protocols, in particular for material-constrained studies where conventional ribodepletion techniques fail.
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Project description:CRISPR/Cas9-targeted removal of unwanted sequences from small-RNA sequencing libraries
| PRJNA476858 | ENA
Project description:A decade since its invention, single-cell RNA sequencing (scRNA-seq) has become a mainstay technology for profiling transcriptional heterogeneity in individual cells. Yet, most existing scRNA-seq methods capture only polyadenylated mRNA to avoid expending resources on profiling the types of transcripts that are usually not-of-interest, such as ribosomal RNA (rRNA). Hence, protocols that enable analysis of the whole transcriptome remain scarce. We adapted a method called DASH (Depletion of Abundant Sequences by Hybridisation) for rRNA depletion from single-cell total RNA-seq libraries. Our analyses show that with our single-cell DASH (scDASH), rRNAs are effectively depleted with minimal non-specificity. Importantly, the rest of the transcriptome is significantly enriched for detection as a result of liberated sequencing quota from depleted rRNA.
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Project description:Most of small RNA library construction methods are based on RNA ligases, which prefer to join the molecules (small RNAs and adapters) that can anneal to each other and form a ligase favoured structure. Different platforms for next generation sequencing use different adapter sequences, causing the cloning bias. Adapters with degenerated nucleotides at the ligating ends (High Definition, HD adapters) were developed to reduce the cloning bias. However, above 90% of the cloning products is adapter dimer when the current available commercial kits and their corresponding protocols are used. Here we adopted and further improved a method demonstrated in a publically available patent (http://www.google.com/patents/WO2011056866A2?cl=en). Using the improved method, we constructed the small RNA libraries by using the total RNA of chondrosarcoma cell line. The adapter dimer was significantly reduced. The small RNA sequences were also analysed. The small RNA libraries of cultured chondrosarcoma cell line were constructed by using an improved protocol where high-definition (HD) adapters were used.
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Project description:Most of small RNA library construction methods are based on RNA ligases, which prefer to join the molecules (small RNAs and adapters) that can anneal to each other and form a ligase favoured structure. Different platforms for next generation sequencing use different adapter sequences, causing the cloning bias. Adapters with degenerated nucleotides at the ligating ends (High Definition, HD adapters) were developed to reduce the cloning bias. However, above 90% of the cloning products is adapter dimer when the current available commercial kits and their corresponding protocols are used. Here we adopted and further improved a method demonstrated in a publically available patent (http://www.google.com/patents/WO2011056866A2?cl=en). Using the improved method, we constructed the small RNA libraries by using the total RNA of Medicago truncatula leaf tissue. The adapter dimer was significantly reduced. The small RNA sequences were also analysed. The small RNA libraries of medicago truncatula leaves were constructed by using an improved protocol where high-definition (HD) adapters were used.
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Project description:MicroRNA (miRNA) and other types of small regulatory RNAs play a crucial role in the regulation of gene expression in eukaryotes. Several distinct classes of small regulatory RNAs have been discovered in recent years. To extend the repertoire of small regulatory RNAs characterized in chickens we used a deep sequencing approach developed by Solexa (now Illumina Inc.). We sequenced three small RNA libraries prepared from different developmental stages of the chicken embryo (days 5, 7, and 9) to produce over 9.5 million short sequence reads. We developed a bioinformatics pipeline to distinguish authentic mature miRNA sequences from other classes of small RNAs and short RNA fragments represented in the sequencing data. Using this approach we detected almost all of the previously known chicken miRNAs and their respective miRNA* sequences. In addition we discovered 449 putative new chicken miRNAs. Of these, 430 miRNAs appear to be specific to the avian lineage. Another 6 new miRNAs had evidence of evolutionary conservation in at least one vertebrate species outside of the bird lineage. The remaining 13 putative miRNAs appear to represent chicken orthologs of known vertebrate miRNAs. We discovered 39 additional putative miRNA candidates originating from miRNA generating intronic sequences known as mirtrons. Keywords: miRNA discovery, mirtrons, chicken embryo
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