Oct 19, 2017

Public workspaceFACS sorting and genome amplification of single cells V.1

DOI
dx.doi.org/10.17504/protocols.io.kbzcsp6
  • Claudia Bergin1,
  • Anna-Maria Divne1
  • 1The Microbial Single Cell facility, SciLifelab, Uppsala, Sweden
  • SINGEK_ITN
Anna-Maria Divne
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DOI: dx.doi.org/10.17504/protocols.io.kbzcsp6
External link: https://www.scilifelab.se/facilities/single-cell/
Protocol Citation: Claudia Bergin, Anna-Maria Divne 2017. FACS sorting and genome amplification of single cells. protocols.io https://dx.doi.org/10.17504/protocols.io.kbzcsp6
Manuscript citation:
(1).Rinke C, Lee J, Nath N, Goudeau D, Thompson B, Poulton N, Dmitrieff E, Malmstrom R, Stepanauskas R, Woyke T. Nat Protoc. 2014 May;9(5):1038-48. doi: 10.1038/nprot.2014.067. Epub 2014 Apr 10. PMID: 24722403
(2).REPLI-g ® Single Cell Handbook, Sample & Assay Technologies, Qiagen 2012.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: October 17, 2017
Last Modified: March 23, 2018
Protocol Integer ID: 8281
Keywords: Single Cell FACS sorting, Single Cell Genomics
Abstract
This protocol describes fluorescence-activated single cell sorting using a MoFlo Astrios EQ cell sorter (Beckman Coulter) and whole genome amplification of single sorted cells in plate formats using the REPLI-g Single Cell kit (Qiagen).
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Guidelines
General cleanroom rules
FACS sorting and whole genome amplification is performed in a cleanroom. Protective clothing such as lab coats, shoe covers, are required for entering the room and additional protection with gloves, hair nets and mouth covers are needed when the plates are sorted into and further processed. Lab coats should be dedicated to a specific task/analysis i.e. sorting or whole genome amplification. The screening of the amplified material is done outside the cleanroom in a PCR hood dedicated to DNA work. To keep a clean environment, take off your gloves when using the computer or touch screen. For a more detailed description of the different steps and critical issues see the paper by Rinke et al 2014 (1).
Whole genome amplification
Whole genome amplification is performed as described for the “amplification of genomic DNA from single cells” in the REPLI-g Single Cell Handbook (2), with the exception that volumes are scaled down with a quarter of the original volume, hence 12.5 µl in total.
Lysis and neutralization reagents from the REPLI-g Single Cell kit are first added to each well of the sorted plate to facilitate genome amplification. The MDA master mix is then added, the plate is sealed with an optical sealing and the reaction is monitored every 15 minutes in real-time at 30 °C for 8 h using a DNA-binding dye (SYTO13) and an instrument suitable for fluorescent readings e.g. a PCR machine or a plate reader. Note that the REPLI-g Single Cell kit mastermix should not be UV-decontaminated as it is already treated to minimize contamination. Tubes, water, lysis and stop solution can however be UVed for 2 Joules before use.
I. Fluorescence-activated cell sorting for single cell genomics
I. Fluorescence-activated cell sorting for single cell genomics
General
FACS sorting and whole genome amplification is performed in a cleanroom. Protective clothing such as lab coats, shoe covers, are required for entering the room and additional protection with gloves, hair nets and mouth covers are needed when the plates are sorted into and further processed. Lab coats should be dedicated to a specific task/analysis i.e. sorting or whole genome amplification. The screening of the amplified material is done outside the cleanroom in a PCR hood dedicated to DNA work. To keep a clean environment, take off your gloves when using the computer or touch screen. For a more detailed description of the different steps and critical issues see the paper by Rinke et al 2014 (1).
Sort plate preparation
Sort plate preparation
  • 1 µl of sterile filtered 1x PBS is pipetted to each well of a 96-well plate. The plate is covered by a clear sealing and then UV-treated for 2 Joule.
  • Prepared plates are kept at 4°C until sorting the same day.
Staining of sample
Staining of sample
  • Thaw the sample on ice.
  • Run the sample through a 70 µm cell strainer to get rid of cell aggregates.
  • Run the unstained sample for sample analysis.
  • Depending on the event rate (eps = events per second), the sample is diluted accordingly.
  • Add SYBR Green I to a final concentration of 1x to the sample.
  • Stain the sample in the dark and on ice for at least 20 min.
  • During analysis of the stained samples the region of interest for sorting are chosen.
  • Define the sort layout in the CyClone plate layout. Always include a few empty wells as negative controls and a few wells with more than 1 cell (e.g 10 cells) as positive controls.
  • Clean the sort chamber and the work space with a decontamination solution, e.g. DNA AWAY®.
Running a clean single cell sort
Running a clean single cell sort
  • Take one plate, carefully remove the sealing.
  • Place the plate into the sort chamber, pay attention that the plate is positioned correctly.
  • Start the sample, then start the sort.
  • Once the sort is finished, carefully take out the plate and immediately cover it with a sealing. Spin down, and transfer the plate to the freezer.
  • Continue with the next plate.
  • Sorted plates are kept at -20°C for short-term, or -80°C for long-term storage.
II. Cell lysis and whole genome amplification using MDA
II. Cell lysis and whole genome amplification using MDA
Whole genome amplification is performed as described for the “amplification of genomic DNA from single cells” in the REPLI-g Single Cell Handbook (2), with the exception that volumes are scaled down with a quarter of the original volume, hence 12.5 µl in total.
Lysis and neutralization reagents from the REPLI-g Single Cell kit are first added to each well of the sorted plate to facilitate genome amplification. The MDA master mix is then added, the plate is sealed with an optical sealing and the reaction is monitored every 15 minutes in real-time at 30 °C for 8 h using a DNA-binding dye (SYTO13) and an instrument suitable for fluorescent readings e.g. a PCR machine or a plate reader. Note that the REPLI-g Single Cell kit mastermix should not be UV-decontaminated as it is already treated to minimize contamination. Tubes, water, lysis and stop solution can however be UVed for 2 Joules before use.
General cleanroom rules and cleaning procedure
General cleanroom rules and cleaning procedure
  • Always wear shoe covers when entering the cleanroom.
  • Use a dedicated lab coat, and only wear it inside the cleanroom, use it at all times.
  • Use hair net and mouth cover when you set up the MDA reaction.
  • Use fresh gloves each time you re-enter the cleanroom, use gloves for every single step.
  • Wipe all working areas and pipets with DNA AWAY®.
  • Clean your gloves and everything that goes into the MDA hood with DNA AWAY®.
General considerations
General considerations
To minimize contamination
  • Change tips between every well (or rows/columns if you use a multichannel pipette).
  • Mix and spin down all reagents before use to avoid possible drops in the lid coming in contact with your gloves.
  • Open sorted plates only in the PCR hood.
  • Include negative controls such as “no sort controls” and no template controls if possible, to keep track of potential contamination.
Reagent handling
  • Keep all reagents on ice after thawing or preparation. The polymerase should be kept in a cooling block.
  • After adding the nucleic binding dye SYTO13, protect the MDA mastermix from light using foil.
Preparation for MDA
Preparation for MDA
  • Clean the hood working area with DNA AWAY®.
  • Wipe pipettes with DNA AWAY®.
  • Wipe all equipment that you will need, including enough pipette tips, with DNA AWAY® and place them in the hood.
  • Turn on the UV in the hood for 20 to 30 min.
  • Fill an ice-box with ice and place a lid wrapped in foil on top of it, fill this lid with ice (will be used for the UV-linker).
  • Fill an ice-box with ice for the reagents later on.
Prepare the lysis, stop solution and MDA master mix
Prepare the lysis, stop solution and MDA master mix
  • Thaw plates on ice in an ice box
  • Add lysis buffer (Buffer D2) in an 8-well strip and close with an 8-cap lid. Do the same for the stop solution.
  • Mix all reagents for the MDA, including the SYTO13 dye, and distribute it in an 8-well strip and close with an 8-cap lid. Protect the reaction from light.
Lysis and neutralization
Lysis and neutralization
  • Add 0,75 µl lysis reagents on the rim of the wells in each row using a multichannel pipette.
  • Close the plate with a sealing and spin down.
  • Put the plate in a thermocycler at 65° C for 10 min.
  • Add 0,75 µl stop solution on the rim of the in each row using a multichannel pipette.
  • Close the plate with sealing and spin down.
MDA
MDA
  • Add 10 µl of MDA-mix reagents on the rim of the wells in each row using a multichannel pipette.
  • Close the plate with a sealing and spin down.
  • Put the plate in a qPCR machine or a plate reader and incubate the plate at 30 °C for 8 h while reading the fluorescence every 15 minutes.
  • The reaction is inactivated at 3 min 65 °C
Screening of amplified material
Screening of amplified material
Screening of whole genome amplification (WGA) products is done using the ROCHE lightCycler 480 and the ROCHE kit LightCycler 480 SYBR Green I Master in 10 ul reactions. Dilute PCR products x 100 and add 2 ul of the DNA to the PCR-mix including the forward and reverse primer.  Add a melting curve analysis after the PCR cycling for evaluation of positive reactions by comparing Tm-values of the samples and positive as well as negative controls.
Purification of PCR-products
Purification of PCR-products
Purified PCR-products or WGA can be quantified in a plate format using the Quant-IT PicoGreen® dsDNA Assay kit (Thermo Fisher Scientific) and a plate reader as described in the REPLI-g Single Cell handbook from Qiagen Appendix B (2).