Protocol for single-cell isolation and genome amplification of environmental microbial eukaryotes for genomic analysis

Journal Article · 12 January 2022 · Star Protocols

DOI: https://doi.org/10.1016/j.xpro.2021.100968 · OSTI ID:1895337

Ciobanu, Doina ^[1]; Chan, Sandy  ^[2]; Ahrendt, Steven ^[3]; Quandt, C. Alisha ^[4]; Benny, Gerald L. ^[5]; Smith, Matthew E.  ^[5]; James, Timothy Y. ^[4]; Cheng, Jan-Fang  ^[1]

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1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Joint Genome Institute (JGI)
2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Joint Genome Institute (JGI); Geisel School of Medicine at Dartmouth, Hanover, NH (United States)
3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Joint Genome Institute (JGI); Univ. of California, Berkeley, CA (United States)
4. Univ. of Michigan, Ann Arbor, MI (United States)
5. Univ. of Florida, Gainesville, FL (United States)

We describe environmental microbial eukaryotes (EMEs) sample collection, single-cell isolation, lysis, and genome amplification, followed by the rDNA amplification and OTU screening for recovery of high-quality species-specific genomes for de novo assembly. These protocols are part of our pipeline that also includes bioinformatic methods. The pipeline and its application on a wide range of phyla of different sample complexity are described in our complementary paper. In addition, this protocol describes optimized lysis, genome amplification, and OTU screening steps of the pipeline. For complete details on the use and execution of this protocol, please refer to Ciobanu et al. (2021).

• Research Organization: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
• Sponsoring Organization: National Science Foundation (NSF); USDOE Office of Science (SC), Biological and Environmental Research (BER)
• Grant/Contract Number: AC02-05CH11231
• OSTI ID: 1895337
• Journal Information: Star Protocols, Journal Name: Star Protocols Journal Issue: 1 Vol. 3; ISSN 2666-1667
• Publisher: Cell PressCopyright Statement
• Country of Publication: United States
• Language: English