TITLE: Isolating Plasmodium sexual and asexual parasites from malaria infected individuals for single cell RNA sequencing
AUTHORS: Sunil Kumar Dogga, Antoine Dara, Jesse Rop, Abdoulaye Djimdé, Arthur Talman, Mara Lawniczak

[DESCRIPTION]
This protocol isolates circulating sexual and asexual Plasmodium parasites from an infected individual's blood such that the resulting parasites are viable for single cell RNA sequencing, as well as bulk RNA sequencing and genome sequencing.
The important features of the protocol include (i) preventing the activation of gametocytes by re-suspending the blood in Suspended Animation (SA) buffer, (ii)  using MACS columns to concentrate capture of circulating gametocytes and late stages from blood, (iii) using Plasmodipur filters to eliminate human contamination from white blood cells, and (iv) performing selective erythrocyte lysis using Streptolysin O (SLO) to remove as many uninfected erythrocytes as possible and improve the percentage of infected erythrocytes for further analysis. 

We have successfully used this protocol on parasites captured from the blood of naturally infected carriers in Mali to generate single cell RNA sequencing data on both asexual and sexual parasites of P. falciparum, P. ovale and P. malariae using the 10X 3' protocol. Because both the asexual stage parasites and gametocytes of P. malariae and P. ovale spp. circulate in the peripheral blood, we recover asexual stages in both the MACS and non-MACS fractions for these two species. 

Human contamination from high quantity transcripts present in erythrocytes remains a problem as SLO is not always successful at removing the majority of uninfected erythrocytes. This contributes to a significant proportion of sequencing output, and decision to sequence at required depth should be taken upon inspection of the RBC-specific transcript peak (~700 nt) post-cDNA amplification QC.  In our experience, when there is a significant RBC-specific peak, we recommend not proceeding with single cell RNA sequencing unless at least 3000 cells can be targeted for recovery.

[BEFORE_START]
Preparation
Pre-cool QuadroMACS Separation Unit and MACS columns (keep them in the fridge the day before the experiment)
Ice bucket
Keep required amount of SA buffer in 50 mL falcons (filled to the top) on ice
Keep centrifuge for 15 ml/50 ml falcons at 4°C

Activate SLO
Resuspend Streptolysin O from Streptococcus Pyrogenes (#S5265-25KU, from Sigma Aldrich) in 5mL of D-PBS and make 150 µl aliquots of 5 units/µl each.
 Prepare 50 µl aliquots of 20mM DTT solution from 1M DTT stock solution (with RNAse free water)
Activate SLO by mixing 150 µl SLO solution (5 units/µl) with 50 µl 20mM DTT solution and incubate at 37°C for 2 hours (to activate the toxin). Activated aliquots may be stored at -20C

[STEPS]
SECTION: Blood Draw
1. Draw fresh blood into a CPDA vacutainer, mix by inverting 4-5 times and immediately keep on ice (Heparin might inhibit PCR/RT. EDTA affects parasites if left for a few hours).
 

SECTION: Blood Draw
2. Following fresh blood draw or transportation as above, add 5 mL  of cold SA buffer to  3 mL  of subject blood sample in a 15mL falcon and keep it on ice

SECTION: Purification of circulating gametocytes and late stage parasites.
3. Take a cooled MACS LS column and insert it into the precooled magnet. Place a labeled 50 mL falcon  beneath it for flow-through.
 

SECTION: Purification of circulating gametocytes and late stage parasites.
4. Pre-wet the MACS LS column with 800 µL of cold SA buffer onto the MACS LS column.
SECTION: Purification of circulating gametocytes and late stage parasites.
5. Decant the sample from Step 2 on the MACS LS column and let it elute.
SECTION: Purification of circulating gametocytes and late stage parasites.
6. Washing the MACS column to elute uninfected erythrocytes and rings (e.g. cells not bound magnetically):
SECTION: Purification of circulating gametocytes and late stage parasites.
6.2. Once the eluate solution is clear, wash with an extra 6 mL of SA buffer. 

SECTION: Purification of circulating gametocytes and late stage parasites.
7. Spin the flow through at 500 x g, 3 min, 4 °C and keep on ice after the spin is complete. This is the fraction that contains early asexuals. (This fraction is processed according to section "Purification of circulating early asexual stages" from Step 12)

8. Elute the MACS fraction:
8.2. Add another 6 mL of SA buffer to the MACS column and insert plunger contained in the MACS column packet and elute by applying gentle (important) pressure.

8.3. Spin down the MACS fraction at 800 x g, 5 min, 4 °C


 




SECTION: Purification of circulating gametocytes and late stage parasites.
6.3. Optional: Wash again with an extra 6 mL of SA buffer.
 


8.4. Remove all but ~120 µL of SA buffer and resuspend the pellets gently.
 

9. Resuspend and take a sample of 100 µL and transfer to an 1.5 mL Eppendorf and keep on ice.
 


10. Take 10 µLfrom the rest of the 20 ul and use it to count on a hemocytometer.

11. Spin the rest of the 10 µL at  800 x g, 5 min, 4 °C, remove most of the supernatant, leaving enough to place 2 x ~2 μl drops on a glass slide, let it dry for 10 min and
 stain with Giemsa to estimate parasitemia.

SECTION: Purification of circulating early asexual stages.
12. From the flowthrough fraction (from Step 7), remove the supernatant and any visible buffy coat (WBCs). Wash by resuspending the pellet with 10 mL of D-PBS and spin at 500 x g, 3 min. 

SECTION: Purification of circulating early asexual stages.
13. Remove supernatant and resuspend in 10 mL of D-PBS for an additional wash. Spin again at 500 x g, 3 min 

SECTION: Purification of circulating early asexual stages.
15. During the above two spins, remove the plunger from a 20 mL syringe, mount it onto a Plasmodipur filter and load 2 mL of sterile PBS and pre-rinse it with gentle pressure. Do the same for a second Plasmodipur. (We are going to pass each sample through 2 Plasmodipur filters to get rid of WBCs)

SECTION: Purification of circulating early asexual stages.
16. Now, load the sample from  Step 14 into the syringe as above and filter it through the Plasmodipur into a 50 mL falcon.

SECTION: Purification of circulating early asexual stages.
17. Reload the filtrate through the second pre-rinsed Plasmodipur and filter it into a 50 mL falcon.
SECTION: Purification of circulating early asexual stages.
18. Spin down the Plasmodipur flowthrough at  500 x g, 3 min and remove the supernatant. 

SECTION: Purification of circulating early asexual stages.
20. Incubate for 10 min at 37 °C for SLO lysis.
 

SECTION: Purification of circulating early asexual stages.
21. Following SLO lysis, spin 1500 x g, 2 min and remove the supernatant (should show heavy lysis)

SECTION: Purification of circulating early asexual stages.
22. Add 1 mL PBS to the tube (even if a clear pellet is not discernible), spin at  1500 x g, 2 min and discard supernatant (a proteinaceous translucent red layer sits above the actual pellet)

SECTION: Purification of circulating early asexual stages.
23. Repeat washing until the supernatant is clear. A clear pellet might not be discernible after the washed. Proceed anyway.
SECTION: Purification of circulating early asexual stages.
24. Resuspend in ~60 µL of D-PBS and keep on ice.
 


SECTION: Purification of circulating early asexual stages.
25. Take an aliquot of 10 µL to count under a hemocytometer.

SECTION: Purification of circulating early asexual stages.
26. Take an aliquot of 10 µL to spin and smear (either a drop smear or a normal smear depending on the size of the pellet) and stain with Giemsa to estimate parasitemia.

SECTION: Example Application: Sample Loading on Chromium 10x
27. Obtain the number of cells per μL in the isolated fractions using a hemocytometer.

SECTION: Example Application: Sample Loading on Chromium 10x
28. Estimate parasitemia of isolated fractions after staining with Giemsa.
SECTION: Purification of circulating gametocytes and late stage parasites.
6.1. Wash with 4 mL of SA buffer
 


8.1. During the spin above (Step 7), remove the column from the magnet and place into a labeled 15 mL falcon tube. Add 4 mL of SA buffer to the column to elute the cells trapped in the MACS column by gravity.

SECTION: Purification of circulating early asexual stages.
14. Remove supernatant and resuspend pellet in 10 mL of D-PBS
SECTION: Purification of circulating early asexual stages.
19. Take 50 µL from the blood pellet above in a 1.5mL Eppendorf tube and resuspend in 950 µL PBS + 100 µL activated SLO.
29. Calculate the number of infected RBCs per μL by using the cell number and parasitemia estimate. 


 

30. Follow the guidelines from Chromium 10x User Guide to target required recovery of cells.