TITLE: Blood sampling, cell isolation, single-cell GEM-generation, globin mRNA blockers and sequencing library preparation protocol
AUTHORS: Dorien De Pooter, Ben De Clerck, Koen Dockx, Domenica De Santis, Sarah Sauviller, Pascale Dehertogh, Matthias Beyens, Isabelle Bergiers, Isabel Nájera, Ellen Van Gulck, Nádia Conceição-Neto, Wim Pierson

[DESCRIPTION]
This protocol details blood sampling, cell isolation, single-cell GEM-generation, globin mRNA blockers and sequencing library preparation.

[STEPS]
SECTION: Blood sampling
1. Place the animal in the restrainer so that only one of the two the hind legs and tail are free. Stretch out the leg. 
SECTION: Blood sampling
2. Remove the fur from the lateral side of the hind leg using the electric clipper. 
SECTION: Blood sampling
3. Locate the lateral saphenous vein (If necessary: swab the skin with a small amount of ethanol to help visualize the vein).
SECTION: Blood sampling
4. Puncture the vein with the needle at a 90° angle at the most distal visible site. 
SECTION: Blood sampling
5. Collect drops of blood until 200 µLis collected in the respective capillary tube. 

 

SECTION: Blood sampling
6. Use a dry piece of gauze to apply pressure to the puncture site until bleeding stops.
SECTION: Blood sampling
7. Remove the mouse from the restrainer. 
SECTION: Blood sampling
8. Dispose the needle in an approved sharps container.
SECTION: Blood sampling
9. Close the trumpet-shaped side of the tube by pushing the cap.

For leukocyte isolation: add extra 3 µL of EDTA solution to the tube, close the upper cap of the tube and vortex thoroughly.
For serum analysis: close the upper cap of the tube. Do not vortex.
SECTION: Blood sampling
10. Store the tubes at Room temperature until further processing.

SECTION: ACK lysis buffer selection
11. 
 

Pipet the blood out of the capillary tubes and transfer the entire volume of blood, ~150 µL to a 50 mL tube.

SECTION: ACK lysis buffer selection
12. Add 2 mL of ACK buffer (1x) and incubate the sample on a rocker for 5 minat Room temperature.

SECTION: ACK lysis buffer selection
13. Add 10 mL of 1x PBS to the top of the tube to stop the lysis reaction and centrifuge the lysate sample at 400 x g, 5 min.

SECTION: ACK lysis buffer selection
14. Pour off the supernatant. Add10 mLPBS. Gently pipette 10 times to resuspend the pellet.

SECTION: ACK lysis buffer selection
15. Filter the cell suspension through a 70 µm cell strainer and centrifuge at 400 x g, 5 min.

SECTION: ACK lysis buffer selection
16. Pour off the supernatant and add 1 mL of ACK buffer to the pellet and incubate on a rocker for 5 min.

SECTION: ACK lysis buffer selection
17. Add 10 mL of 1x PBS to the top of the tube to stop the lysis reaction and centrifuge the lysate sample at 250 x g, 10 min.

SECTION: ACK lysis buffer selection
18. Pour off the supernatant and perform a second wash with 10 mL of PBS and centrifuge at 250 x g, 10 min.
SECTION: ACK lysis buffer selection
19. Pour off the supernatant and resuspend the pellet in equal volume of PBS-0.04% BSA to original whole blood volume.
SECTION: ACK lysis buffer selection
20. Count cells using the Cellaca or Luna cell counter.
SECTION: Anti-Ter119 Microbeads selection
21. 
 
 
Transfer the blood from the capillary tubes over a 70 µm pluristrainer placed on a 5 mL round-bottom tube to remove any clots. 

SECTION: Anti-Ter119 Microbeads selection
22. Rinse the original blood tubes twice with 100 µL of MACS buffer and rinse the filter with 3 mL MACS buffer.
SECTION: Anti-Ter119 Microbeads selection
23. Centrifugate the samples at 400 x g, 5 min .
SECTION: Anti-Ter119 Microbeads selection
24. Pipet off the supernatant (∼3.3 mL) with a P1000 pipettor.

SECTION: Anti-Ter119 Microbeads selection
25. Briefly vortex the Anti-Ter119 microbeads and add 200 µL of beads. Mix well and incubate for 15 min. 

SECTION: Anti-Ter119 Microbeads selection
26. Add 3.5 mLof MACS buffer, mix the solution by inverting and centrifugate at 300 x g, 10 min.

SECTION: Anti-Ter119 Microbeads selection
27. Prepare the LD columns by putting them in the magnetic field of a QuadroMACS Separator and rinse columns with 2 mL MACS buffer; discard flowthrough and place a new 5 mL round-bottom tube under the columns.

SECTION: Anti-Ter119 Microbeads selection
28. From the centrifugated blood, remove supernatant using a P1000 pipettor and resuspend cells in 1000 µLof MACS buffer.

SECTION: Anti-Ter119 Microbeads selection
29. Apply the cell suspension onto the column and collect the unlabeled cells which pass through.
SECTION: Anti-Ter119 Microbeads selection
30. Wash tube that contained the blood-bead mixture with 1 mL of buffer and add to column. Add an additional 1 mL of MACS buffer to elute all the cells. 

SECTION: Anti-Ter119 Microbeads selection
31. Mix the effluent with cells of interest, by inverting and spin down at 250 x g, 10 min.

SECTION: Anti-Ter119 Microbeads selection
32. Pour the supernatant and resuspend the cells in 4 mL of MACS buffer.
SECTION: Anti-Ter119 Microbeads selection
33. Spin down cells at 400 x g, 5 min. Pour supernatant and resuspend pellet in cell culture media (500 µL) or 150 µL of PBS-0.04% BSA for a total volume of 200 µL.

SECTION: Anti-Ter119 Microbeads selection
34. Count cells using the Cellaca or Luna cell counter.
SECTION: ACK and Anti-Ter119 Microbeads selection
35. 
 

Pipet the blood out of the capillary tubes and transfer the entire volume of blood, ∼150 µL to a 50 mL tube.

SECTION: ACK and Anti-Ter119 Microbeads selection
36. Add 2 mL of ACK buffer (1x) and incubate the sample on a rocker for 10 min .

SECTION: ACK and Anti-Ter119 Microbeads selection
37. Add 4 mL of PBS to the top of the tube to stop the lysis reaction and centrifuge the sample at 400 x g, 5 min.

SECTION: ACK and Anti-Ter119 Microbeads selection
38. Pour off the supernatant and add 4 mL of MACS buffer to wash the cells, centrifuge at 250 x g, 10 min.
SECTION: ACK and Anti-Ter119 Microbeads selection
39. Resuspend the pellet in 540 µL of MACS buffer and add 50 µL of Anti-Ter119 MicroBeads. 

SECTION: ACK and Anti-Ter119 Microbeads selection
40. Mix well and incubate for 15 min.

SECTION: ACK and Anti-Ter119 Microbeads selection
41. After incubation, add 3.5 mL of MACS buffer and centrifuge at 300 x g, 10 min.

SECTION: ACK and Anti-Ter119 Microbeads selection
42. After centrifugation, pour off the supernatant and resuspend cells in 500 µL MACS buffer.

SECTION: ACK and Anti-Ter119 Microbeads selection
43. Prepare the LD columns by putting them in the magnetic field of a QuadroMACS Separator, place a 50 µm cell strainer onto the columns and prepare filter and column by rinsing with 2 mL of MACS buffer; discard flowthrough and place a new 5 mL tube under the columns.

SECTION: ACK and Anti-Ter119 Microbeads selection
44. From the centrifugated blood, take off supernatant using a P1000 pipettor and resuspend cells in 1000 µLof MACS buffer.

SECTION: ACK and Anti-Ter119 Microbeads selection
45. Apply the cell suspension onto the column and collect the unlabeled cells which pass through.
SECTION: ACK and Anti-Ter119 Microbeads selection
46. Wash tube with 1 mL of buffer and add to column. Add an additional 1 mLof MACS buffer to elute all the cells. Collect the total effluent which contains the white blood cells.

SECTION: ACK and Anti-Ter119 Microbeads selection
47. Mix the suspension by inverting and spin the total effluent down at 250 x g, 10 min.
SECTION: ACK and Anti-Ter119 Microbeads selection
48. Pour the supernatant and resuspend the cells in 4 mLof MACS buffer.

SECTION: ACK and Anti-Ter119 Microbeads selection
49. Spin down cells at 400 x g, 5 min. Pour supernatant and resuspend pellet in cell media (500 µL) or 150 µLof PBS-0.04% BSA for a total volume of 200 µL.

SECTION: ACK and Anti-Ter119 Microbeads selection
50. Count cells using the Cellaca or Luna cell counter.
SECTION: Histopaque-1119 selection
51. 
 

Prepare 5 mL tube with 3 mL Histopaque-1119 gradient fresh on the day of the experiment.


SECTION: Histopaque-1119 selection
52. Pipet the blood out of the capillary tubes and slowly add it to the top of the gradient, and centrifuge the tube for 400 x g, 30 min with low deceleration for 10 min.

SECTION: Histopaque-1119 selection
53. Remove, slowly, the white ring of cells at the top of the gradient.
SECTION: Histopaque-1119 selection
54. Spin down cells at 400 x g, 5 min.

SECTION: Histopaque-1119 selection
55. Resuspend cells in 200 µL of PBS-0.04% BSA.

SECTION: Histopaque-1119 selection
56. Count cells using the Cellaca or Luna cell counter.
SECTION: Library preparation and sequencing
57. Evaluate cell viability and cell counts with acridine orange/propidium iodine (AO/PI) onto the Luna FX7 cell counter (Logos Biosystem).
SECTION: Library preparation and sequencing
58. Adjust the volume of the cell suspension to 1000 cells/µL in PBS-0.04% BSA. 
SECTION: Library preparation and sequencing
59. For each sample, load 20,000 cells (supplementary table 1) into the Chromium Controller (10X Genomics) and partition into single 10X barcoded droplet according to the Chromium single cell 3’ Gel Bead Kit v2 manufacturer’s instructions. 
SECTION: Library preparation and sequencing
59.1. For a selection of samples that underwent anti-Ter 119 Microbeads isolation protocol, use a modified master mix recipe: 1 µL of 0.1X FastSelect globin mRNA blockers 1X (QIAseq FastSelect – Globin Kit, Qiagen GmbH) was introduced at the expense of 1 µL of water according to manufacturer’s instructions. 

SECTION: Library preparation and sequencing
59.2. For a selection of other samples, use a modified GEM-RT incubation on a Veriti 96- well thermal cycler (Thermofisher), as shown below: 


 

SECTION: Library preparation and sequencing
60. For the single-cell gene expression libraries, use the Chromium Single Cell 3’ Reagent kit v2 (10X Genomics) according to manufacturer’s instructions. 
SECTION: Library preparation and sequencing
60.1. cDNAs recovered are amplified on Veriti 96-Well Thermal Cycler, the cDNAs products are amplified, cleaned up, quality controlled and quantified. 
SECTION: Library preparation and sequencing
61. Add Illumina P5, P7, Read2 primers and Sample Index to the cDNAs products to generate sequencing libraries. 
SECTION: Library preparation and sequencing
62. Perform quality control and quantify using a quantitative PCR (KAPA Biosystems Library Quantification Kit for Illumina platforms). 
SECTION: Library preparation and sequencing
63. Load the sequencing libraries on a NextSeq2000 P3 flow cell with a sequencing depth of 50,000 reads per cell.