RNA sequencing library construction for Illumina GA II v1

protocols.io ◽  
2015 ◽  
Author(s):  
Jian Cao ◽  
Julie Ni ◽  
Wenxiu Ma ◽  
Vanessa Shiu ◽  
Luis A ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Library Construction ◽  
Sequencing Library

scholarly journals Excess primer degradation by Exo I improves the preparation of 3′ cDNA ligation-based sequencing libraries

BioTechniques ◽  
2019 ◽  
Vol 67 (3) ◽  
pp. 110-116
Author(s):  
Christel Hougård Enroth ◽  
Annaleigh Ohrt Fehler ◽  
Line Dahl Poulsen ◽  
Jeppe Vinther
Keyword(s):  
Escherichia Coli ◽  
Rna Sequencing ◽  
Small Rna ◽  
Reverse Transcription ◽  
Library Construction ◽  
Reverse Transcription Primer ◽  
Adapter Ligation ◽  
Sequencing Library

RNA sequencing library construction using single-stranded ligation of a DNA adapter to 3′ ends of cDNAs often produces primer–adapter byproducts, which compete with cDNA–adapter ligation products during library amplification and, therefore, reduces the number of informative sequencing reads. We find that Escherichia coli Exo I digestion efficiently and selectively removes surplus reverse transcription primer and thereby reduces the primer–adapter product contamination in 3′ cDNA ligation-based sequencing libraries, including small RNA libraries, which are typically similar in size to the primer–adapter products. We further demonstrate that Exo I treatment does not lead to trimming of the cDNA 3′ end when duplexed with the RNA template. Exo I digestion is easy to perform and implement in other protocols and could facilitate a more widespread use of 3′ cDNA ligation for sequencing-based applications.

scholarly journals Bias in Ligation-Based Small RNA Sequencing Library Construction Is Determined by Adaptor and RNA Structure

PLoS ONE ◽  
2015 ◽  
Vol 10 (5) ◽  
pp. e0126049 ◽  
Author(s):  
Ryan T. Fuchs ◽  
Zhiyi Sun ◽  
Fanglei Zhuang ◽  
G. Brett Robb
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
Rna Structure ◽  
Small Rna Sequencing ◽  
Library Construction ◽  
Sequencing Library

scholarly journals Methyltransferase-directed orthogonal tagging and sequencing of miRNAs and bacterial small RNAs

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Milda Mickutė ◽  
Kotryna Kvederavičiūtė ◽  
Aleksandr Osipenko ◽  
Raminta Mineikaitė ◽  
Saulius Klimašauskas ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Regulatory Networks ◽  
Library Preparation ◽  
Rna Seq ◽  
Basic Principles ◽  
Cofactor Binding ◽  
Sequencing Library ◽  
Sequencing Library Preparation ◽  
Target Rna

Abstract Background Targeted installation of designer chemical moieties on biopolymers provides an orthogonal means for their visualisation, manipulation and sequence analysis. Although high-throughput RNA sequencing is a widely used method for transcriptome analysis, certain steps, such as 3′ adapter ligation in strand-specific RNA sequencing, remain challenging due to structure- and sequence-related biases introduced by RNA ligases, leading to misrepresentation of particular RNA species. Here, we remedy this limitation by adapting two RNA 2′-O-methyltransferases from the Hen1 family for orthogonal chemo-enzymatic click tethering of a 3′ sequencing adapter that supports cDNA production by reverse transcription of the tagged RNA. Results We showed that the ssRNA-specific DmHen1 and dsRNA-specific AtHEN1 can be used to efficiently append an oligonucleotide adapter to the 3′ end of target RNA for sequencing library preparation. Using this new chemo-enzymatic approach, we identified miRNAs and prokaryotic small non-coding sRNAs in probiotic Lactobacillus casei BL23. We found that compared to a reference conventional RNA library preparation, methyltransferase-Directed Orthogonal Tagging and RNA sequencing, mDOT-seq, avoids misdetection of unspecific highly-structured RNA species, thus providing better accuracy in identifying the groups of transcripts analysed. Our results suggest that mDOT-seq has the potential to advance analysis of eukaryotic and prokaryotic ssRNAs. Conclusions Our findings provide a valuable resource for studies of the RNA-centred regulatory networks in Lactobacilli and pave the way to developing novel transcriptome and epitranscriptome profiling approaches in vitro and inside living cells. As RNA methyltransferases share the structure of the AdoMet-binding domain and several specific cofactor binding features, the basic principles of our approach could be easily translated to other AdoMet-dependent enzymes for the development of modification-specific RNA-seq techniques.

High-Throughput Illumina Strand-Specific RNA Sequencing Library Preparation

2011 ◽  
Vol 2011 (8) ◽  
pp. pdb.prot5652-pdb.prot5652 ◽  
Author(s):  
S. Zhong ◽  
J.-G. Joung ◽  
Y. Zheng ◽  
Y.-r. Chen ◽  
B. Liu ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
High Throughput ◽  
Library Preparation ◽  
Sequencing Library ◽  
Sequencing Library Preparation

scholarly journals Nanoliter-scale next-generation sequencing library-mediated high-throughput 16S rRNA microbial community profiling

BioTechniques ◽  
2020 ◽  
Vol 68 (4) ◽  
pp. 204-210
Author(s):  
Hui Zhang ◽  
Xiangdan Yu ◽  
Zhe Zhang ◽  
Zhenhua Liu ◽  
Cong Tang ◽  
...  
Keyword(s):  
Microbial Community ◽  
Next Generation Sequencing ◽  
Large Scale ◽  
Amplicon Sequencing ◽  
Next Generation ◽  
Library Construction ◽  
Microbial Community Profiling ◽  
Community Profiling ◽  
Sequencing Library ◽  
Generation Sequencing

An ultra-high-throughput workflow for next-generation sequencing library construction at nanoliter scale for amplicon sequencing, termed Smartchip Nanowell Platform for Target Enrichment, was established using a nanodispenser system and a nanoliter-scale PCR chip. To demonstrate its cost and time advantages over conventional methods for library construction, quality control and pooling for large-scale samples, target amplicon sequencing of the 16S ribosomal RNA gene V3-V4 region widely used for microbial community profiling was chosen for comparison. The finding of no significant difference in microbial community profiling between the two methods strongly supports the conclusion that Smartchip Nanowell Platform for Target Enrichment is a cost-effective method for next-generation sequencing library construction for large-scale samples to conduct amplicon sequencing-based applications.

scholarly journals Miniaturization and optimization of 384-well compatible RNA sequencing library preparation

PLoS ONE ◽  
2019 ◽  
Vol 14 (1) ◽  
pp. e0206194 ◽  
Author(s):  
Madeline Y. Mayday ◽  
Lillian M. Khan ◽  
Eric D. Chow ◽  
Matt S. Zinter ◽  
Joseph L. DeRisi
Keyword(s):  
Rna Sequencing ◽  
Library Preparation ◽  
Sequencing Library ◽  
Sequencing Library Preparation

scholarly journals A Novel in vitro Model for Prostate Cancer Bone Metastasis using Microfluidics

2020 ◽  
Vol 1 (2) ◽  
Author(s):  
Alexander Cui ◽  
Et al.
Keyword(s):  
Prostate Cancer ◽  
Bone Metastasis ◽  
Rna Sequencing ◽  
Cell Types ◽  
Pcr Analysis ◽  
Bone Microenvironment ◽  
Sequencing Library ◽  
Multiple Cell ◽  
Vitro Model

Alexander Cui, Srinivasa Rao, Sam Olechnowicz, James Edwards, Peter Cook, Edmond Walsh, Claire Edwards Prostate cancer (PCa) cells predominantly metastasise to bone, and once this happens the disease is incurable. Hence it is vital to understand the mechanism of bone metastasis in PCa. Several models have been established to study PCa bone metastasis in vitro, involving the co-culture of PCa cell lines with cells of the bone microenvironment such as osteoblasts, osteoclasts, bone marrow stromal cells, etc. However, these models are limited to one-to-one co-culture (i.e., PCa cells co-cultured with one other cell type), which overly simplifies the complex bone microenvironment consisting of multiple cell types. We aimed to overcome this limitation by adapting a microfluidics platform that employs a novel mechanism to isolate sub-microliter volumes using fluid walls. By using this technology, we were able to successfully differentiate osteoblasts and adipocytes from precursor cells (2T3 and ST2 cells respectively) in nanowells, and co-cultured them with PC3 prostate cancer cells. Differentiation to osteoblasts and adipocytes was confirmed by calcein green and BODIPY live staining which could be monitored using the Incucyte imaging platform. Following co-culture, the PCa cells were trypsinised and total RNA was extracted from them. A cDNA amplification step was successfully incorporated into the reverse transcription protocol to provide sufficient material for quantitative PCR analysis. Further, sequencing adapters were added to the cDNA to generate an RNA-sequencing library for transcriptome-wide analysis. Using this novel model, with imaging, RNA-sequencing and qPCR as read-outs, we are working on elucidating the signalling pathways that are activated by the bone microenvironment in PCa cells. 

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