TITLE: Single-cell Whole Genome Amplification (scWGA) of human frozen post-mortem brain samples isolated by Laser Capture Microdissection (LCM)
AUTHORS: Ester Kalef-Ezra, Amy Bowes, Christos Proukakis

[DESCRIPTION]
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).

[STEPS]
SECTION: Section 1: Tissue cryo-sectioning
1. UV pre-treat PEN slides and pre-label them with a pencil prior use.
 

SECTION: Section 1: Tissue cryo-sectioning
2. Clean and UV the cryostat prior use and set the temperature approx. @-20 °C.

SECTION: Section 1: Tissue cryo-sectioning
3. Place a new blade on cryostat.
 

SECTION: Section 1: Tissue cryo-sectioning
4. Transfer human post-mortem frozen tissue chunks with dry-ice to the cryostat.
SECTION: Section 1: Tissue cryo-sectioning
5. Embed the tissue in OCT embedding medium. 
 

SECTION: Section 1: Tissue cryo-sectioning
6. Mount the cryo-block onto the specimen clamp.
SECTION: Section 1: Tissue cryo-sectioning
7. Trim the tissue to get a plane surface.
SECTION: Section 1: Tissue cryo-sectioning
8. Cut thick sections of tissue (14 mm) and immediately place the cryo-sectioned tissue onto the pre-labelled PEN membrane glass slides.
SECTION: Section 1: Tissue cryo-sectioning
9. Keep the slides in a box on dry ice during the until all cryo-sectioning procedure is completed for the slides of interest.
SECTION: Section 1: Tissue cryo-sectioning
10. Use slides immediately or store the slides @-80 °C in tightly sealed containers.

SECTION: Section 2: Giemsa staining
11. Prepare Giemsa working solution: 10 Giemsa (stock) + 90 mL Buffer pH pH 6.8, Mix well and leave to stand for 10 min @Room temperature.
 

SECTION: Section 2: Giemsa staining
12. Thaw slides gradually, e.g., from @-80 °C → 2 min @-20 °C → 2 min @4 °C → 2 min @Room temperature.

SECTION: Section 2: Giemsa staining
13. Immerse slides in a jar containing ice-cold Methanol for 5 min @Room temperature.

SECTION: Section 2: Giemsa staining
14. Quick (5 s) air-dry the slides. 
 

SECTION: Section 2: Giemsa staining
15. Immerse slides in a jar containing Giemsa working solution for150 s @Room temperature.

SECTION: Section 2: Giemsa staining
16. Rinse slides with DNase free water.
SECTION: Section 2: Giemsa staining
17. Air-dry the slides for at least 15 min @Room temperature.

SECTION: Section 2: Giemsa staining
18. Proceed directly to LCM. 
 
 
SECTION: Section 3: Laser Capture Microdissection (LCM)
19. Prepare fresh Chromatin Digestion Buffer (1 millimolar (mM) EDTA, 20 millimolar (mM) TRIS pH pH 8.0, 0.2 mg/mL Proteinase K, 0.001% Triton X, in nuclease free water) as on Keinath et al 2015.

SECTION: Section 3: Laser Capture Microdissection (LCM)
20. UV collecting tubes for at least 30 min prior to use.
SECTION: Section 3: Laser Capture Microdissection (LCM)
21. Clean the LCM with 70% EtOH and DNase Away prior use.
SECTION: Section 3: Laser Capture Microdissection (LCM)
22. Capture single nuclei using the LCM guidelines. 
 
 
 

SECTION: Section 3: Laser Capture Microdissection (LCM)
24. Add 10 µL of Chromatin Digestion Buffer on the cap and close the tube carefully.

SECTION: Section 3: Laser Capture Microdissection (LCM)
25. Vortex tubs for at least 5 s.

SECTION: Section 3: Laser Capture Microdissection (LCM)
26. Spin down for 5 min @Room temperature and place on ice until further use.

SECTION: Section 3: Laser Capture Microdissection (LCM)
27. Incubate tubes @55 °C in an oven O/N.

SECTION: Section 4: Single-cell Whole Genome Amplification (scWGA)
28. 
 
After O/N incubation @55 °C in an oven O/N: 
SECTION: Section 4: Single-cell Whole Genome Amplification (scWGA)
28.1. Quick spin.
SECTION: Section 4: Single-cell Whole Genome Amplification (scWGA)
28.2. Heat treat: @75 °C for 10 min → @95 °C for 4 min → @4 °C hold.
SECTION: Section 4: Single-cell Whole Genome Amplification (scWGA)
28.3. Quick spin and transfer samples on ice.

SECTION: Section 4: Single-cell Whole Genome Amplification (scWGA)
28.4. Follow PicoPlex protocol steps skipping the lysis step.
SECTION: Section 3: Laser Capture Microdissection (LCM)
23. 
 
Use Zeiss 415190-9181-000 collection tubes for cutting. After cut:
SECTION: Section 3: Laser Capture Microdissection (LCM)
24. Observe the nucleus on the cap in the LCM microscope.
SECTION: Section 3: Laser Capture Microdissection (LCM)
25. Transfer tubes in a stereoscope.
SECTION: Section 3: Laser Capture Microdissection (LCM)
26. While observing the caps under the stereoscope add 5 µL of Chromatin Digestion Buffer on the caps and pipette multiple times upside/down to release the nucleus from the cap.

SECTION: Section 3: Laser Capture Microdissection (LCM)
27. Transfer the nucleus with the buffer to the bottom of the tubes.
SECTION: Section 3: Laser Capture Microdissection (LCM)
28. Repeat the process by adding other 5 µL of Chromatin Digestion Buffer on the caps and ensure that all sample has been transferred to the bottom of the tubes.

SECTION: Section 3: Laser Capture Microdissection (LCM)
29. Close the tube.
SECTION: Section 3: Laser Capture Microdissection (LCM)
30. Spin down for 5 min @Room temperature and place the tube in the oven @55 °C in an oven O/N.

SECTION: Section 3: Laser Capture Microdissection (LCM)
31. Repeat for all samples.
SECTION: Section 4: Single-cell Whole Genome Amplification (scWGA)
32. 
 
After O/N incubation @55 °C in an oven O/N: 

SECTION: Section 4: Single-cell Whole Genome Amplification (scWGA)
32.1. Quick spin.
SECTION: Section 4: Single-cell Whole Genome Amplification (scWGA)
32.2. Heat treat: @75 °C for 10 min → @95 °C for 4 min → @4 °C hold.

SECTION: Section 4: Single-cell Whole Genome Amplification (scWGA)
32.3. Quick spin and transfer samples on ice.
SECTION: Section 4: Single-cell Whole Genome Amplification (scWGA)
32.4. Follow PicoPlex protocol steps skipping the lysis step.
SECTION: Section 3: Laser Capture Microdissection (LCM)
23. Observe the nucleus on the cap in the Leica LCM microscope.