Similar Datasets
Project description:This experiment was designed to improve the proportion of miRNA mapping reads from sRNA library sequencing, by reducing the amount of particularly abundant rRNA fragments.
2018-02-25 | GSE98832 | GEO
Project description:Reduction of rRNA during sRNA library construction using blocking oligos
| PRJNA386331 | ENA
Project description:This experiment was designed to improve the percentage of miRNA mapping reads from sRNA library sequencing, by reducing the amount of one particularly abundant rRNA 30mer sequence.
2018-02-25 | GSE86313 | GEO
Project description:Balancing protein expression is critical when optimizing genetic systems. Typically, this requires library construction to vary the genetic parts controlling each gene, which can be expensive and time-consuming. Here, we develop sRNAs corresponding to 15nt 'target' sequences that can be inserted upstream of a gene. The targeted gene can be repressed from 1.6- to 87-fold by controlling sRNA expression using promoters of different strength. A pool is built where six sRNAs are placed under the control of 16 promoters that span a ?103-fold range of strengths, yielding ?107 combinations. This pool can simultaneously optimize up to six genes in a system. This requires building only a single system-specific construct by placing a target sequence upstream of each gene and transforming it with the pre-built sRNA pool. The resulting library is screened and the top clone is sequenced to determine the promoter controlling each sRNA, from which the fold-repression of the genes can be inferred. The system is then rebuilt by rationally selecting parts that implement the optimal expression of each gene. We demonstrate the versatility of this approach by using the same pool to optimize a metabolic pathway (?-carotene) and genetic circuit (XNOR logic gate).
| S-EPMC5737548 | biostudies-literature
Project description:Genetic screens using single-guide-RNA (sgRNA) libraries and CRISPR technology have been powerful to identify genetic regulators for both coding and noncoding regions of the genome. Interrogating functional elements in noncoding regions requires sgRNA libraries that are densely covering, and ideally inexpensive, easy to implement and flexible for customization. We present a Molecular Chipper protocol for generating dense sgRNA libraries from genomic regions of interest. This approach utilizes a combination of random fragmentation and a Type III restriction enzyme to derive a dense coverage of sgRNA library from input DNA.
| S-EPMC5564686 | biostudies-other
Project description:CRISPR/Cas9-based gene knockout libraries have emerged as a powerful tool for functional screens. We present here a set of pre-designed human and mouse sgRNA sequences that are optimized for both high on-target potency and low off-target effect. To maximize the chance of target gene inactivation, sgRNAs were curated to target both 5΄ constitutive exons and exons that encode conserved protein domains. We describe here a robust and cost-effective method to construct multiple small sized CRISPR library from a single oligo pool generated by array synthesis using parallel oligonucleotide retrieval. Together, these resources provide a convenient means for individual labs to generate customized CRISPR libraries of variable size and coverage depth for functional genomics application.
| S-EPMC5499874 | biostudies-literature
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.
2015-01-31 | E-GEOD-59330 | biostudies-arrayexpress
Project description:BackgroundRNA sequencing has become the gold standard for transcriptome analysis but has an inherent limitation of challenging quantification of low-abundant transcripts. In contrast to microarray technology, RNA sequencing reads are proportionally divided in function of transcript abundance. Therefore, low-abundant RNAs compete against highly abundant - and sometimes non-informative - RNA species.ResultsWe developed an easy-to-use strategy based on high-affinity RNA-binding oligonucleotides to block reverse transcription and PCR amplification of specific RNA transcripts, thereby substantially reducing their abundance in the final sequencing library. To demonstrate the broad application potential of our method, we applied it to different transcripts and library preparation strategies, including YRNAs in small RNA sequencing of human blood plasma, mitochondrial rRNAs in both 3' end sequencing and long-read sequencing, and MALAT1 in single-cell 3' end sequencing. We demonstrate that the blocking strategy is highly efficient, reproducible, specific, and generally results in better transcriptome coverage and complexity.ConclusionOur method does not require modifications of the library preparation procedure apart from simply adding blocking oligonucleotides to the RT reaction and can thus be easily integrated into virtually any RNA sequencing library preparation protocol.
| S-EPMC9996952 | biostudies-literature
Project description:BackgroundCell-free DNA (cfDNA), present in circulating blood plasma, contains information about prenatal health, organ transplant reception, and cancer presence and progression. Originally developed for the genomic analysis of highly degraded ancient DNA, single-stranded DNA (ssDNA) library preparation methods are gaining popularity in the field of cfDNA analysis due to their efficiency and ability to convert short, fragmented DNA into sequencing libraries without altering DNA ends. However, current ssDNA methods are costly and time-consuming.ResultsHere we present an efficient ligation-based single-stranded library preparation method that is engineered to produce complex libraries in under 2.5 h from as little as 1 nanogram of input DNA without alteration to the native ends of template molecules. Our method, called Single Reaction Single-stranded LibrarY or SRSLY, ligates uniquely designed Next-Generation Sequencing (NGS) adapters in a one-step combined phosphorylation/ligation reaction that foregoes end-polishing. Using synthetic DNA oligos and cfDNA, we demonstrate the efficiency and utility of this approach and compare with existing double-stranded and single-stranded approaches for library generation. Finally, we demonstrate that cfDNA NGS data generated from SRSLY can be used to analyze DNA fragmentation patterns to deduce nucleosome positioning and transcription factor binding.ConclusionsSRSLY is a versatile tool for converting short and fragmented DNA molecules, like cfDNA fragments, into sequencing libraries while retaining native lengths and ends.
| S-EPMC6935139 | biostudies-literature
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.
2015-01-31 | GSE59330 | GEO