Dec 07, 2023

Public workspaceSingle-cell Whole Genome Amplification (scWGA) of human frozen post-mortem brain samples isolated by Laser Capture Microdissection (LCM)

DOI
dx.doi.org/10.17504/protocols.io.5qpvorjw7v4o/v1
  • Ester Kalef-Ezra1,2,
  • Amy Bowes3,4,
  • Christos Proukakis1,2
  • 1Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, London, UK;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815;
  • 3Cancer Genomics Group, The Francis Crick Institute, London, UK;
  • 4Sarcoma Biology and Genomics Group, UCL Cancer Institute, London, UK
  • University College London
Ester Kalef-Ezra
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DOI: dx.doi.org/10.17504/protocols.io.5qpvorjw7v4o/v1
Protocol Citation: Ester Kalef-Ezra, Amy Bowes, Christos Proukakis 2023. Single-cell Whole Genome Amplification (scWGA) of human frozen post-mortem brain samples isolated by Laser Capture Microdissection (LCM). protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvorjw7v4o/v1
Manuscript citation:
REFERENCES:
Part of this protocol was adapted from the publication:
Keinath MC, Timoshevskiy VA, Timoshevskaya NY, Tsonis PA, Voss SR, Smith JJ. Initial characterization of the large genome of the salamander Ambystoma mexicanum using shotgun and laser capture chromosome sequencing. Sci Rep. 2015 Nov 10;5:16413. doi: 10.1038/srep16413. PMID: 26553646; PMCID: PMC4639759
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
We use this protocol and it's working
Created: June 01, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82740
Keywords: Giemsa, Laser Capture Microdissection, LCM, Single-cell, Whole Genome Amplification, WGA, brain, ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Grant ID: 000430
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Abstract
This protocol uses Laser Capture Microdissection (LCM) technology on human post-mortem brain tissue slides with rapid Giemsa staining to isolate single nuclei for Single-cell Whole Genome Amplification (scWGA) in order to do low coverage (<1x) single-cell whole genome sequencing to detect mega-base somatic Copy Number Variations (CNVs).
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Materials
Materials:

  • OCT Mounting media (VWR 361603E)
  • Cryochem Cryostat disinfectant (Solmedia REA023)
  • PEN Membrane Slide (Thikness4 µm ) glass (Leica 11600288)
  • Collection tubes
o For Leica LCM: Amount0.2 mL MaxyClear thin-walled flat cap Maximum Recovery PCR tube (Axygen AXY2050)
o For Zeiss LCM: AdhesiveCap 200 opaque (Zeiss 415190-9181-000)
  • Microtome blades compatible with the cryostat to be used (e.g., Feather S35 type)
  • Giemsa (Sigma #1092040100)
  • Buffer pH 6.8 (Merck #111374): Dissolve 1 tablet in Amount1000 mL in Water and store @TemperatureRoom temperature
Note
Note: This Buffer is stable for up to 4 weeks.

  • Ultra-Pure DNase/RNase-Free Distilled Water (Thermo Fisher 10977049)
  • 70% EtOH for surface cleaning
  • DNA AWAY Surface Decontaminant (Thermo Scientific 7010PK)
  • Cleaning wipes (e.g., Conti Washcloth Dry Brosch Direct PH5959)
  • brush, pencil, slide jars, slide box
  • PicoPlex (Takara R300671, R300672, R300673)

Equipment:

  • Cryostat (we use Bright Instruments OTF6000)
  • Laser Capture Microdissection (we have used Leica LMD7000 and Zeiss Microbeam PALM Laser Capture Microscope system)
  • UV source (UV stratalinker or hood with a UV lamp)



Section 1: Tissue cryo-sectioning
Section 1: Tissue cryo-sectioning
UV pre-treat PEN slides and pre-label them with a pencil prior use.
Note
Note: Use a PCR or cell culture hood with UV lab and UV the slides for 30 min. If available, a UV stratalinker can be used.

Clean and UV the cryostat prior use and set the temperature approx. @Temperature-20 °C .

Temperature
Place a new blade on cryostat.
Note
Caution! Be careful because the blade is sharp.

Note: Change blade when cutting samples from different donors or brain regions to avoid contamination.

Critical
Transfer human post-mortem frozen tissue chunks with dry-ice to the cryostat.
Embed the tissue in OCT embedding medium.
Note
Note: Use minimal amounts OCT, as it will affect the cryo-sectioning process.

Mount the cryo-block onto the specimen clamp.
Trim the tissue to get a plane surface.
Cut thick sections of tissue (14 mm) and immediately place the cryo-sectioned tissue onto the pre-labelled PEN membrane glass slides.
Keep the slides in a box on dry ice during the until all cryo-sectioning procedure is completed for the slides of interest.
Use slides immediately or store the slides @Temperature-80 °C in tightly sealed containers.

Temperature
Section 2: Giemsa staining
Section 2: Giemsa staining
Prepare Giemsa working solution: 10 Giemsa (stock) + Amount90 mL Buffer pH Ph6.8 , Mix well and leave to stand for Duration00:10:00 @TemperatureRoom temperature .
Note
Caution! Work with Giemsa under chemical hood and store it in a glass bottle.

10m
Mix
Critical
Thaw slides gradually, e.g., from @Temperature-80 °C → Duration00:02:00 @Temperature-20 °C → Duration00:02:00 @Temperature4 °C → Duration00:02:00 @TemperatureRoom temperature .

6m
Temperature
Immerse slides in a jar containing ice-cold Methanol for Duration00:05:00 @TemperatureRoom temperature .

5m
Quick (Duration00:00:05 ) air-dry the slides.
Note
Note: If needed, at this stage remove excess OCT with a brush.

5s
Immerse slides in a jar containing Giemsa working solution forDuration00:02:30 @TemperatureRoom temperature .

2m 30s
Rinse slides with DNase free water.
Wash
Air-dry the slides for at least Duration00:15:00 @TemperatureRoom temperature .

15m
Proceed directly to LCM.
Note
Note: Stained slides can be stored in the fridge and used later for cutting but single-cell whole genome amplification efficiency may be reduced.
Image 1: Example of human cingulate cortex tissue image on a PEN slide stained with Giemsa.
Section 3: Laser Capture Microdissection (LCM)
Section 3: Laser Capture Microdissection (LCM)
Prepare fresh Chromatin Digestion Buffer (Concentration1 millimolar (mM) EDTA, Concentration20 millimolar (mM) TRIS pH Ph8.0 , Amount0.2 mg/mL Proteinase K, 0.001% Triton X, in nuclease free water) as on Keinath et al 2015.

UV collecting tubes for at least 30 min prior to use.
Clean the LCM with 70% EtOH and DNase Away prior use.
Capture single nuclei using the LCM guidelines.
Note
We have used 2 different LCM technologies and cells were cut in 20x magnification while keeping laser power to a minimum. It is advisable to take images prior and after cutting of the slide, and also on the cap after cutting for each single cell.
Image 2: Examples of a single nuclei prior cut and after cut on the slide, as well as after cut on the cap.
Note
Note 1: To minimize contamination, cut closely to the nuclei of interest.

Note 2: Use at least one negative control, which could be an empty collection tube that had been placed on the collector, but additional controls could include:
  • Collection tube with a micro-dissected area of the slide that does not contain a nucleus.
  • An Empty collection tube not opened outside PCR hood.
  • Collection tube with a micro-dissected area of the slide which contains a nucleus that is only partly cut.

Step case

Option A
10 steps

Leica Laser Capture Microscope system LMD system
Observe the nucleus on the cap in the Leica LCM microscope.
Add Amount10 µL of Chromatin Digestion Buffer on the cap and close the tube carefully.

Pipetting
Vortex tubs for at least Duration00:00:05 .

5s
Mix
Spin down for Duration00:05:00 @TemperatureRoom temperature and place TemperatureOn ice until further use.

5m
Centrifigation
Incubate tubes @Temperature55 °C in an oven O/N.

Incubation
Section 4: Single-cell Whole Genome Amplification (scWGA)
Section 4: Single-cell Whole Genome Amplification (scWGA)
14m
14m

Note
Note: We found PicoPlex amplification with typical PicoPlex lysis steps for LCM samples was suboptimal, hence our lysis is more extensive.
After O/N incubation @Temperature55 °C in an oven O/N:
Quick spin.
Heat treat: @Temperature75 °C for Duration00:10:00 → @Temperature95 °C for Duration00:04:00 → @Temperature4 °C hold.
14m
Temperature
Quick spin and transfer samples TemperatureOn ice .

Follow PicoPlex protocol steps skipping the lysis step.