scholarly journals Comparison of Poly-A+ Selection and rRNA Depletion in Detection of lncRNA in Two Equine Tissues Using RNA-seq

2020 ◽  
Vol 6 (3) ◽  
pp. 32 ◽  
Author(s):  
Anna R. Dahlgren ◽  
Erica Y. Scott ◽  
Tamer Mansour ◽  
Erin N. Hales ◽  
Pablo J. Ross ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Preparation Method ◽  
Parietal Lobe ◽  
Library Preparation ◽  
Rna Seq ◽  
The Past ◽  
Rrna Depletion ◽  
Non Coding Rnas ◽  
Nucleolar Rna ◽  
Library Preparation Method

Long non-coding RNAs (lncRNAs) are untranslated regulatory transcripts longer than 200 nucleotides that can play a role in transcriptional, post-translational, and epigenetic regulation. Traditionally, RNA-sequencing (RNA-seq) libraries have been created by isolating transcriptomic RNA via poly-A+ selection. In the past 10 years, methods to perform ribosomal RNA (rRNA) depletion of total RNA have been developed as an alternative, aiming for better coverage of whole transcriptomic RNA, both polyadenylated and non-polyadenylated transcripts. The purpose of this study was to determine which library preparation method is optimal for lncRNA investigations in the horse. Using liver and cerebral parietal lobe tissues from two healthy Thoroughbred mares, RNA-seq libraries were prepared using standard poly-A+ selection and rRNA-depletion methods. Averaging the two biologic replicates, poly-A+ selection yielded 327 and 773 more unique lncRNA transcripts for liver and parietal lobe, respectively. More lncRNA were found to be unique to poly-A+ selected libraries, and rRNA-depletion identified small nucleolar RNA (snoRNA) to have a higher relative expression than in the poly-A+ selected libraries. Overall, poly-A+ selection provides a more thorough identification of total lncRNA in equine tissues while rRNA-depletion may allow for easier detection of snoRNAs.

Transcriptome-wide m6A detection with DART-Seq

2019 ◽  
Author(s):  
Kate D. Meyer
Keyword(s):  
Gene Expression Regulation ◽  
Preparation Method ◽  
Current Knowledge ◽  
Cross Reactivity ◽  
High Variability ◽  
Library Preparation ◽  
Rna Seq ◽  
Generation Sequencing ◽  
Library Preparation Method

Abstract m6A is the most abundant internal mRNA modification and plays diverse roles in gene expression regulation. Much of our current knowledge about m6A has been driven by recent advances in the ability to detect this mark transcriptome-wide. Antibody-based approaches have been the method of choice for global m6A mapping studies. These methods rely on m6A antibodies to immunoprecipitate methylated RNAs, followed by next-generation sequencing to identify m6A-containing transcripts1,2. While these methods enabled the first identification of m6A sites transcriptome-wide and have dramatically improved our ability to study m6A, they suffer from several limitations. These include requirements for high amounts of input RNA, costly and time-consuming library preparation, high variability across studies, and m6A antibody cross-reactivity with other modifications. Here, we describe DART-Seq (deamination adjacent to RNA modification targets), an antibody-free method for global m6A detection. In DART-Seq, the C to U deaminating enzyme, APOBEC1, is fused to the m6A-binding YTH domain. This fusion protein is then introduced to cellular RNA either through overexpression in cells or with in vitro assays, and subsequent deamination of m6A-adjacent cytidines is then detected by RNA sequencing to identify m6A sites. DART-Seq can successfully map m6A sites throughout the transcriptome using as little as 10 nanograms of total cellular RNA, and it is compatible with any standard RNA-seq library preparation method.

scholarly journals Lasy-Seq: a high-throughput library preparation method for RNA-Seq and its application in the analysis of plant responses to fluctuating temperatures

2018 ◽  
Author(s):  
Mari Kamitani ◽  
Makoto Kashima ◽  
Ayumi Tezuka ◽  
Atsushi J. Nagano
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Preparation Method ◽  
Low Cost ◽  
Cost Effective ◽  
Plant Responses ◽  
Library Preparation ◽  
Rna Seq ◽  
Temperature Responses ◽  
Library Preparation Method

AbstractRNA-Seq is a whole-transcriptome analysis method used to research biological mechanisms and functions; its use in large-scale experiments is limited by costs and labour. In this study, we established a high-throughput and cost effective RNA-Seq library preparation method that did not require mRNA enrichment. The method adds unique index sequences to samples during reverse transcription (RT) that is conducted at a higher temperature (≥62°C) to suppress RT of A-rich sequences in rRNA, and then pools all samples into a single tube. Both single-read and paired end sequencing of libraries is enabled. We found that the pooled RT products contained large amounts of RNA, mainly rRNA, and caused over-estimations of the quantity of DNA, resulting in unstable tagmentation results. Degradation of RNA before tagmentation was necessary for the stable preparation of libraries. We named this protocol low-cost and easy RNA-Seq (Lasy-Seq), and used it to investigate temperature responses in Arabidopsis thaliana. We analysed how sub-ambient temperatures (10–30°C) affected the plant transcriptomes, using time-courses of RNA-Seq from plants grown in randomly fluctuating temperature conditions. Our results suggest that there are diverse mechanisms behind plant temperature responses at different time scales.

scholarly journals SPlinted Ligation Adapter Tagging (SPLAT), a novel library preparation method for whole genome bisulphite sequencing

2016 ◽  
Vol 45 (6) ◽  
pp. e36-e36 ◽  
Author(s):  
Amanda Raine ◽  
Erika Manlig ◽  
Per Wahlberg ◽  
Ann-Christine Syvänen ◽  
Jessica Nordlund
Keyword(s):  
Preparation Method ◽  
Whole Genome ◽  
Library Preparation ◽  
Bisulphite Sequencing ◽  
Library Preparation Method

scholarly journals Biogenesis and Function of Small Non-Coding RNAs Derived from Eukaryotic Ribosomal RNA

2018 ◽  
Author(s):  
Marine Lambert ◽  
Abderrahim Benmoussa ◽  
Patrick Provost
Keyword(s):  
Ribosomal Rna ◽  
Potential Role ◽  
A Priori ◽  
Biological Significance ◽  
Rna Seq ◽  
New Class ◽  
Scientific Mind ◽  
Non Coding Rnas ◽  
And Function ◽  
Rrna Sequences

The advent of RNA-sequencing (RNA-Seq) technologies has markedly improved our knowledge and expanded the compendium of small non-coding RNAs, most of which derive from the processing of longer RNA precursors. In this review article, we will discuss about the biogenesis and function of small non-coding RNAs derived from eukaryotic ribosomal RNA (rRNA), called rRNA fragments (rRFs), and their potential role(s) as regulator of gene expression. This relatively new class of ncRNAs remained poorly investigated and underappreciated until recently, due mainly to the a priori exclusion of rRNA sequences—because of their overabundance—from RNA-Seq datasets. The situation surrounding rRFs resembles that of microRNAs (miRNAs), which used to be readily discarded from further analyses, for more than five decades, because we could not believe that RNA of such a short length could bear biological significance. As if we had not yet learned our lesson not to restrain our investigative, scientific mind from challenging widely accepted beliefs or dogmas, and from looking for the hidden treasures in the most unexpected places.

scholarly journals An optimized ribodepletion approach for C. elegans RNA-sequencing libraries

2021 ◽  
Author(s):  
Alec Barrett ◽  
Rebecca McWhirter ◽  
Seth R Taylor ◽  
Alexis Weinreb ◽  
David M Miller ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Noncoding Rnas ◽  
Rrna Gene ◽  
Library Preparation ◽  
C Elegans ◽  
Pcr Duplicates ◽  
Rrna Depletion ◽  
Dissociated Cells ◽  
Probe Set ◽  
Long Genes

ABSTRACTA recent and powerful technique is to obtain transcriptomes from rare cell populations, such as single neurons in C. elegans, by enriching dissociated cells using fluorescent sorting. However, these cell samples often have low yields of RNA that present challenges in library preparation. This can lead to PCR duplicates, noisy gene expression for lowly expressed genes, and other issues that limit endpoint analysis. Further, some common resources, such as sequence specific kits for removing ribosomal RNA, are not optimized for non-mammalian samples. To optimize library construction for such challenging samples, we compared two approaches for building RNAseq libraries from less than 10 nanograms of C. elegans RNA: SMARTSeq V4 (Takara), a widely used kit for selecting poly-adenylated transcripts; and SoLo Ovation (Tecan Genomics), a newly developed ribodepletion-based approach. For ribodepletion, we used a custom kit of 200 probes designed to match C. elegans rRNA gene sequences. We found that SoLo Ovation, in combination with our custom C. elegans probe set for rRNA depletion, detects an expanded set of noncoding RNAs, shows reduced noise in lowly expressed genes, and more accurately counts expression of long genes. The approach described here should be broadly useful for similar efforts to analyze transcriptomics when RNA is limiting.

scholarly journals A novel use of random priming-based single-strand library preparation for whole genome sequencing of formalin-fixed paraffin-embedded tissue samples

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Emily A Saunderson ◽  
Ann-Marie Baker ◽  
Marc Williams ◽  
Kit Curtius ◽  
J Louise Jones ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Preparation Method ◽  
Degraded Dna ◽  
Whole Genome ◽  
Library Preparation ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed ◽  
Library Preparation Method

Abstract The desire to analyse limited amounts of biological material, historic samples and rare cell populations has collectively driven the need for efficient methods for whole genome sequencing (WGS) of limited amounts of poor quality DNA. Most protocols are designed to recover double-stranded DNA (dsDNA) by ligating sequencing adaptors to dsDNA with or without subsequent polymerase chain reaction amplification of the library. While this is sufficient for many applications, limited DNA requires a method that can recover both single-stranded DNA (ssDNA) and dsDNA. Here, we present a WGS library preparation method, called ‘degraded DNA adaptor tagging’ (DDAT), adapted from a protocol designed for whole genome bisulfite sequencing. This method uses two rounds of random primer extension to recover both ssDNA and dsDNA. We show that by using DDAT we can generate WGS data from formalin-fixed paraffin-embedded (FFPE) samples using as little as 2 ng of highly degraded DNA input. Furthermore, DDAT WGS data quality was higher for all FFPE samples tested compared to data produced using a standard WGS library preparation method. Therefore, the DDAT method has potential to unlock WGS data from DNA previously considered impossible to sequence, broadening opportunities to understand the role of genetics in health and disease.

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