Single-cell RNA extraction and cDNA library preparation from challenging marine protists (e.g. Acantharea) v1 (protocols.io.mvhc636)

protocols.io ◽  
2018 ◽  
Author(s):  
Margaret Brisbin ◽  
Lisa Mesrop
Keyword(s):  
Single Cell ◽  
Cdna Library ◽  
Rna Extraction ◽  
Library Preparation ◽  
Marine Protists ◽  
Cdna Library Preparation

cDNA library preparation from total RNA extracts of Single-cell marine protists (e.g. Acantharia, Strombidium basimorphum, and Prymnesium parvum) for transcriptome sequencing v2

2022 ◽  
Author(s):  
Joost S S. Mansour ◽  
Konstantinos Anestis ◽  
Fabrice Not ◽  
Uwe John
Keyword(s):  
Single Cell ◽  
Cdna Library ◽  
Mammalian Cells ◽  
Single Cells ◽  
Rna Extraction ◽  
Cdna Libraries ◽  
Prymnesium Parvum ◽  
Large Variability ◽  
Total Rna ◽  
Marine Protists

Many marine protists are not culturable and therefore challenging to study, nonetheless, they are essential in all marine ecosystems. The development of single-cell techniques is allowing for more marine protists to be studied. Such genomic approaches aim to help to disentangle heterotrophic processes such as phagotrophy from osmotrophy and phototrophic-induced anabolic activities. This information will then support cellular and metabolic modeling by better elucidating the physiological mechanisms and quantifying their importance in different scenarios. However, single-cell protocols and low input RNA kits for transcriptomics are usually made for and tested with mammalian cells, as such the feasibility and efficiency of single-cell transcriptomics on highly diverse mixotrophic protists is not always known. Often single-cell transcriptomics of microbial eukaryotes shows low transcript recovery rates and large variability. We report on transcriptomic methods that we have successfully performed on single cells of Acantharia, Strombidium basimorphum, and Prymnesium parvum. This protocol follows up after total RNA extraction (from the protocol at dx.doi.org/10.17504/protocols.io.bp6xmrfn) to prepare cDNA libraries for Illumina sequencing. The described protocol uses the SMART-Seq4 kit (Takara #634891) for cDNA synthesis and amplification, but this can also be successfully performed with the NEBNext kit (NEB #E6421). The NEBNext kit protocol is very similar to the protocol described here and generally the manufacture's protocol can be followed but see the notes at step 4 and step 18 of this protocol, and do the final elution after cDNA purification in 10 mM Tris (pH 8.0). The subsequent cDNA library is prepared following the .

scholarly journals Nanopore native RNA sequencing of a human poly(A) transcriptome: RNA extraction, cDNA conversion and direct RNA and cDNA library preparation for Oxford Nanopore

2019 ◽  
Author(s):  
Rachael E. Workman ◽  
Alison D. Tang ◽  
Paul S. Tang ◽  
Miten Jain ◽  
John R. Tyson ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Rna Extraction ◽  
Human Cell Line ◽  
Cdna Libraries ◽  
Library Preparation ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Sequencing Strategy ◽  
Oxford Nanopore Technologies ◽  
Cdna Library Preparation

Abstract High throughput cDNA sequencing technologies have dramatically advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and because modifications are not carried forward in cDNA. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies (ONT). Our study focused on poly(A) RNA from the human cell line GM12878, generating 9.9 million aligned sequence reads. These native RNA reads had an aligned N50 length of 1294 bases, and a maximum aligned length of over 21,000 bases. A total of 78,199 high-confidence isoforms were identified by combining long nanopore reads with short higher accuracy Illumina reads. We describe methods for extracting intact RNA, poly-A selection, cDNA conversion for a portion of sample, and library preparation for both direct RNA and cDNA libraries.

cDNA Library Preparation

2013 ◽  
pp. 29-40
Author(s):  
Maarten Kooiker ◽  
Gang-Ping Xue
Keyword(s):  
Cdna Library ◽  
Library Preparation ◽  
Cdna Library Preparation

scholarly journals Barcoded cDNA library preparation for small RNA profiling by next-generation sequencing

Methods ◽  
2012 ◽  
Vol 58 (2) ◽  
pp. 164-170 ◽  
Author(s):  
Markus Hafner ◽  
Neil Renwick ◽  
Thalia A. Farazi ◽  
Aleksandra Mihailović ◽  
John T.G. Pena ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Cdna Library ◽  
Small Rna ◽  
Library Preparation ◽  
Next Generation ◽  
Rna Profiling ◽  
Generation Sequencing ◽  
Cdna Library Preparation
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