title: Improved lysis of single bacterial cells by a modified alkaline-thermal shock procedure.
abstract: Single-cell genomics (SCG) is a recently developed tool to study the genomes of unculturable bacterial species. SCG relies on multiple-strand displacement amplification (MDA), PCR, and next-generation sequencing (NGS); however, obtaining sufficient amounts of high-quality DNA from samples is a major challenge when performing this technique. Here we present an improved bacterial cell lysing procedure that combines incubation in an alkaline buffer with a thermal shock (freezing/heating) treatment to yield highly intact genomic DNA with high efficiency. This procedure is more efficient in lysing Bacillus subtilis and Synechocystis cells compared with two other frequently used lysis methods. Furthermore, 16S ribosomal RNA gene and overall genome recovery were found to be improved by this method using single cells from a Utah desert soil community or Escherichia coli single cells, respectively. The efficiency of genome recovery for E. coli single cells using our procedure is comparable with that of the REPLI-g Single Cell (sc) Kit, but our method is much more economical. By providing high-quality genome templates suitable for downstream applications, our procedure will be a promising improvement for SCG research.
authors.affiliation: Center for Translational Medicine, Department of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, Liaoning, China.|Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM.
authors.forename: Jian
authors.initials: J
authors.lastname: He
authors.affiliation: Engineering Laboratory of Specialty Fibers and Nuclear Energy Materials, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo, Zhejiang, China.
authors.forename: Shiyu
authors.initials: S
authors.lastname: Du
authors.affiliation: Key Laboratory of Biotechnology and Pharmaceutical Engineering, Molecular Pharmacology Research Center, School of Pharmacy, Wenzhou Medical University, Wenzhou, Zhejiang, China.
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Tan
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM.
authors.forename: Ayesha
authors.initials: A
authors.lastname: Arefin
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM.
authors.forename: Cliff S
authors.initials: CS
authors.lastname: Han
mesh_terms: D000863:Antacids / Q000737:chemistry*; D001419:Bacteria / Q000737:chemistry* / Q000235:genetics*; D002458:Cell Fractionation / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry* / Q000302:isolation & purification*; D013696:Temperature
chemical_list: D000863:Antacids; D004269:DNA, Bacterial
keywords: cell lysis; genome recovery; genome sequencing; single-cell genomics; unculturable microbiology