Effective Lysis of Cyanobacterial and Green Algal Single Cells for Whole Genome Amplification in Microfluidics

Summary

Here we present a protocol to lyse cyanobacteria and green algae single cells that allows for subsequent single-cell whole genome amplification in a microfluidic platform with a 100% success rate.

Abstract

Single-cell sequencing is becoming popular for analyzing the genome of a single cell within a heterogenous cell population. It often relies on microfluidic tools to perform single cell isolation and nanoscale chemical reactions to lyse the cell and amplify its genome. However, single-cell sequencing has mainly been applied to human cells and certain bacterial species that are easy to lyse. It is still rare to use single-cell sequencing in environmental studies, as many species of vital environmental significance such as cyanobacterial and green algal species have complex and rigid cell wall structures. To extend single-cell sequencing to these hard-to-lyse species, it is essential to develop an effective lysis method compatible with microfluidic tools and amplification chemistry. Here, we present a lysis method proven effective for cyanobacterial and green algal species for subsequent microfluidic-based single-cell whole genome sequencing (SC-WGA). The protocol combines thermal and chemical lysis mechanisms and has achieved >25 ng DNA for 100% of the single cells after on-chip amplification. Nostoc was chosen as a cyanobacterial model for the protocol development. The optimized protocol was directly applied to Gloeocapsa, another cyanobacterial species, and Sphaerocystis, a eukaryotic green alga, without modifications and achieved a 100% success rate.

Introduction

Single-cell whole genome sequencing (SC-WGS) has been popular for studying the genetic heterogeneity of complex cell communities on a cellular level^1,2,3. Generally, SC-WGS requires single-cell isolation, lysis, and amplifying the femtograms to picograms of genomic DNA to generate enough DNA for standard library preparation (>25 ng)^4,5. Microfluidics is an ideal tool for SC-WGA, as it handles nanoscale of fluid precisely^6,7,8

Protocol

1. Preparation of desiccated cell species (Nostoc, Gloeocapsa, Sphaerocystis)

1. Add 300 µL of sample diluent [0.08% poloxamer 407 in phosphate-buffered saline (PBS)] to 2 mL tubes that contain desiccated samples of Nostoc, Gloeocapsa, and Sphaerocystis, respectively, to re-suspend the cells.
   NOTE: The desiccated samples can be identified visually in the tube as particles before re-suspension and are aggregated to the bottom of the tube after addi.......

Discussion

During the process of single cell isolation in a microfluidic device using laser tweezers, it is essential to ensure that no undesired cells are in the cell isolation chambers prior to adding lysis buffers. Undesired cells should be moved out of the chamber to minimize contaminating DNA caused by these cells. Earlier studies have shown that lasers with wavelength between 1250 nm and 1550 nm with 100 mW power can increase the temperature and rupture the cell membrane⁴⁴. However, other evidence has .......

Materials

Name	Company	Catalog Number	Comments
DTT	Bio-rad	1610611
EDTA	Thermo Fisher	15575020	pH 8.0
Lysozyme	Epicentre	R1804M
MATLAB			microfluidic user interface
Microscope	Nikon		Eclipse/Ti
Nuclease-free water	Thermo Fisher	AM9938
Optical Tweezers	Thorlabs	OTM 211
PBS	Thermo Fisher	10010023	pH 7.4
PDMS	Dow Coring	Sylgard 184
Pluronic F-127	Sigma Aldrich	9003116
Single cell WGA kit	Qiagen	150343	Include D2 buffer and neutralization buffer
Tapestation	Agilent	2200
Tween 20	Sigma Aldrich	9005-64-5