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URL: https://dx.doi.org/DOI:10.17504/protocols.io.hdmb246

Authors: Lisa Mesrop, Sarah Hu

Group: Caron Lab - Protistan Ecology

Summary: Protocol for PCR amplification of extracted cDNA or DNA and downstream library preparation for MiSeq sequencing.See other related protocols for extraction of RNA and/or DNA from environmental samplesThis protocol is specific to 18S rRNA gene tag sequencing targeting to V4 hypervariable region. We use V4 primers with Illumina adapters that then anneal to the P5 & P7 Illumina-specific indices. This barcoding step allows us to multiplex many samples for MiSeq sequencing. The V4 region is ~400bp, so we typically do MiSeq 250x250bp PE sequencing. 

Associated Publications: Boeuf D, Edwards BR, Eppley JM, Hu SK, Poff KE, Romano AE, Caron DA, Karl DM, DeLong EF, Biological composition and microbial dynamics of sinking particulate organic matter at abyssal depths in the oligotrophic open ocean. Proceedings of the National Academy of Sciences of the United States of America 116(24). doi: 10.1073/pnas.1903080116

Affiliations: University of Southern California, University of Southern California

External URL: https://www.neb.com/products/m0494-q5-hot-start-high-fidelity-2x-master-mix

Version: 1

Publication Date: 2017

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