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

Doina Ciobanu; Sandy Chan; Steven Ahrendt; C Alisha Quandt; Gerald L Benny; Matthew E Smith; Timothy Y James; Jan-Fang Cheng

doi:10.1016/j.xpro.2021.100968

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

STAR Protoc. 2022 Jan 13;3(1):100968. doi: 10.1016/j.xpro.2021.100968. eCollection 2022 Mar 18.

Authors

Doina Ciobanu¹, Sandy Chan^{1

2}, Steven Ahrendt^{1

3}, C Alisha Quandt⁴, Gerald L Benny⁵, Matthew E Smith⁵, Timothy Y James⁴, Jan-Fang Cheng¹

Affiliations

¹ US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
² Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA.
³ Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, CA 94720, USA.
⁴ Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI 48109, USA.
⁵ Department of Plant Pathology, University of Florida, Gainesville, FL 32611, USA.

Abstract

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).

Keywords: Cell isolation; Environmental sciences; Flow Cytometry/Mass Cytometry; Genomics; Microbiology; Molecular Biology; Sequence analysis; Sequencing; Single Cell.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Cell Separation
Eukaryota*
Genome
Genomics* / methods