An Effective and Reliable Enzymatic Method for the Depletion of Ribosomal RNA for Preparation of High-Quality Libraries for Transcriptome Sequencing

An Effective and Reliable Enzymatic Method for the Depletion of Ribosomal RNA for Preparation of High-Quality Libraries for Transcriptome Sequencing

7 Jan 2016

Ribosomal RNA (rRNA) accounts for the vast majority of total cellular RNA, and its efficient removal is critical during the RNA-Seq library preparation process in order to maximize the yield of biologically-informative transcriptome-derived reads. Sequencing of total RNA samples that have been depleted of rRNA provides a more comprehensive representation of the transcriptome compared to mRNA sequencing. This application note demonstrates a flexible and highly effective method for targeted enzymatic depletion of rRNA using complementary oligonucleotide probes and RNase H degradation.

KAPA RNA Library Preparation Kits for Illumina

Kapa Biosystems, Inc.

KAPA Stranded mRNA-Seq Kits includes all the enzymes and buffers required for cDNA library preparation for Illumina Next-Generation Sequencing, utilizing 100 ng – 4 µg of total RNA. KAPA mRNA Capture Beads are included for isolation of poly(A)-tailed RNA. Kits provides precise measurement of strand orientation (>99%), uniform coverage, and high-confidence mapping of alternate transcripts, and are optimized for the improved coverage of GC-rich and low-abundance transcripts. Kits contain KAPA HiFi for high-efficiency and low bias library amplification, as well as KAPA mRNA Capture Beads and a streamlined, “with-bead” protocol. KAPA Stranded RNA-Seq Kits include all the enzymes and buffers required for cDNA library preparation for Illumina Next-Generation Sequencing, but do not contain the KAPA mRNA Capture Beads. Kits can be used to prepare libraries from 10-400 ng of either poly(A)-selected, ribosomally-depleted, or total RNA. Features: Uncover challenging transcripts  Improved coverage of GC-rich transcripts Enhanced identification of exonic regions Detect low-abundance transcripts Enables identification of transcripts missed by competitor kits, even with high input High uniformity across varying amounts of sample input Identify more genes Higher percentage of uniquely mapped reads compared to Illumina TruSeq™ Stranded mRNA Sample Prep Kits Lower duplication rates yield better coverage Maintain high coverage uniformity Minimal 5′–3′ bias across transcripts More uniform distribution of reads over each transcript   Applications: Gene expression Single nucleotide variation (SNV) discovery Post-transcriptional SNVs Fusion gene identification Targeted transcriptome Whole transcriptome

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RNA InterferenceRNA interference (RNAi) uses siRNA or miRNA for transcriptional silencing, gene knockdown and regulation of gene expression. RNAi requires chemical synthesis, introduction of DNA vectors into cells, an assay of RNAi effects and RNAi quantification or analysis. Consider target sequence selection, reagent preparation, controls, high specificity and effectiveness and low non-specific gene knockdown.Next Generation SequencingNext-generation sequencing (NGS), also known as whole-genome sequencing, high-throughput sequencing and massive parallel sequencing, produces and analyses thousands to millions of nucleotide sequences at once. Sequencing systems operate via varying technologies depending on the manufacturer, including sequencing by synthesis, ligation, pyrosequencing, ion semiconductor and single-molecule real-time sequencing. For NGS, library preparation is paramount to successful sequencing. In this section, explore a range of library preparation kits, from targeted, amplicon-based or hybridization-based kits including epigenomic, transcriptomic and genomic workflows to fragmentation kits. Find the best next-generation sequencing products in our peer-reviewed product directory: compare products, check customer reviews and receive pricing direct from manufacturers.OligonucleotidesOligonucleotides are small nucleic acid polymers, usually less than 20 bases in length. Oligonucleotides can be made via enzymatic cleavage or more commonly by chemical synthesis with polymerases. Their use includes FISH, southern blots, microarrays and as primers in PCR. High fidelity synthesis kits and detection systems are available for easy production and detection, respectively.Library PreparationLibrary preparation is a critical step in sequencing workflows, where DNA or RNA samples are converted into libraries for high-throughput, next generation sequencing. This step ensures accurate results and minimizes biases. Explore library preparation kits in our peer-reviewed product directory; compare products, check reviews, and get pricing directly from manufacturers.RNARNA is a nucleic acid that plays a key role in gene expression and protein synthesis. It serves as a messenger between DNA and ribosomes, carrying genetic instructions to produce proteins. Advances in RNA-based therapies, such as mRNA vaccines and gene editing, have revolutionized treatment strategies for genetic disorders and infectious diseases. Explore RNA research tools and therapies in our peer-reviewed product directory; compare products, check customer reviews, and get pricing directly from manufacturers.SequencingSequencing refers to determining the order of nucleotides in a DNA or RNA molecule. This process is essential in genomics, diagnostics, and evolutionary biology. Techniques like next-generation sequencing (NGS) and Sanger sequencing provide high-throughput and precise data for analyzing genomes and transcriptomes. Browse our peer-reviewed product directory to find the best sequencing tools and reagents, compare products, check reviews, and get pricing directly from manufacturers.TranscriptomicsEnzymes