cDNA library preparation from total RNA extracts of Single-cell marine protists (e.g. Acantharia, Strombidium basimorphum, and Prymnesium parvum) for transcriptome sequencing v1 (protocols.io.bp7vmrn6)

protocols.io ◽  
2020 ◽  
Author(s):  
Joost Mansour ◽  
Konstantinos Anestis ◽  
Fabrice Not ◽  
Uwe John
Keyword(s):  
Single Cell ◽  
Cdna Library ◽  
Transcriptome Sequencing ◽  
Prymnesium Parvum ◽  
Library Preparation ◽  
Total Rna ◽  
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 .

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

scholarly journals An improved iCLIP protocol

2021 ◽  
Author(s):  
Flora C. Y. Lee ◽  
Anob M. Chakrabarti ◽  
Heike Hänel ◽  
Elisa Monzón-Casanova ◽  
Martina Hallegger ◽  
...  
Keyword(s):  
Cdna Library ◽  
Binding Sites ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Library Preparation ◽  
Powerful Technique ◽  
Uv Crosslinking ◽  
Nucleotide Resolution ◽  
Cdna Library Preparation

AbstractCrosslinking and Immunoprecipitation (CLIP) is a powerful technique to obtain transcriptome-wide maps of in vivo protein-RNA interactions, which are important to understand the post-transcriptional mechanisms mediated by RNA binding proteins (RBPs). Many variant CLIP protocols have been developed to improve the efficiency and convenience of cDNA library preparation. Here we describe an improved individual nucleotide resolution CLIP protocol (iiCLIP), which can be completed within 4 days from UV crosslinking to libraries for sequencing. For benchmarking, we directly compared PTBP1 iiCLIP libraries with the iCLIP2 protocol produced under standardised conditions, and with public eCLIP and iCLIP PTBP1 data. We visualised enriched motifs surrounding the identified crosslink positions and RNA maps of these crosslinks around the alternative exons regulated by PTBP1. Notably, motif enrichment was higher in iiCLIP and iCLIP2 in comparison to public eCLIP and iCLIP, and we show how this impacts the specificity of RNA maps. In conclusion, iiCLIP is technically convenient and efficient, and enables production of highly specific datasets for identifying RBP binding sites.

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