TITLE: isolating human malaria parasites for RNA-sequencing at ultra-low densities
AUTHORS: Kathryn Milne, Florian Bach, Wiebke Nahrendorf, J. Alexandra Rowe, Philip J Spence

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The large subtelomeric multi-gene families encoded in the </span><span style = "font-style:italic;">Plasmodium</span><span> genome play critical roles in parasite virulence, chronicity and transmission. However, quantifying parasite gene expression </span><span style = "font-style:italic;">in vivo</span><span> has been hampered by a lack of sensitive and unbiased RNA-sequencing methodologies that can perform effectively on human blood samples containing small numbers of parasites. The biggest stumbling block is not parasite number per se but host contamination (e.g. white cell transcripts and globin mRNA). These host-derived transcripts outnumber parasite mRNA by many orders of magnitude, preventing their measurement regardless of the depth of sequencing. The solution to this problem is to isolate intact, viable malaria parasites from whole blood and to process these parasites for RNA-sequencing in the absence of host material. Here we present a step-by-step guide to isolating </span><span style = "font-style:italic;">P. falciparum</span><span> and </span><span style = "font-style:italic;">P. vivax</span><span> blood-stage parasites (at any life-cycle stage) from healthy volunteers participating in human challenge studies. We have optimised the effective removal of white cells, the gentle permeabilisation of red cell membranes - leaving the parasitophorous vacuole intact - and maximised the recovery of viable parasites. Crucially, this protocol is highly effective at ultra-low densities (just 5,000 parasites per ml) and has been used to track </span><span style = "font-style:italic;">var</span><span> gene switching </span><span style = "font-style:italic;">in vivo</span><span> and perform PacBio sequencing of new clonal field isolates. Other downstream applications, such as </span><span style = "font-style:italic;">ex vivo</span><span> single cell RNA-sequencing, are possible and this protocol could also be adapted for patient samples from endemic areas and returning travellers. The ability to isolate malaria parasites directly from human blood at ultra-low densities will empower the community to address the role of parasite variant surface antigens in naive and semi-immune hosts.</span></div></div>

[STEPS]
?. [prepare in advance]
securely insert the correct rotor into the centrifuges you will use
?. [leukocyte depletion]
Removing virtually all white blood cells is essential for successful down-stream analysis of the parasite transcriptome. Each white cell contains at least 200 times the amount of RNA compared to a ring-stage parasite and at a density of 5000 parasites*ml-1 white cells outnumber parasites by 1000 to 1. As such, if leukocyte depletion is inefficient it will not be possible to detect parasite transcripts (and increasing the depth of sequencing will not overcome the white cell noise).
?. [prepare in advance]
carefully clean centrifuges as well as your bench and equipment (including pipettes) with 1% chemgene and 70% ethanol
?. [prepare in advance]
set water-bath to 37°C, collect ice & dry ice
?. [prepare in advance]
prepare fresh working saponin solution (57 ml per volunteer) using stock saponin. keep on ice until use (for details see buffers and solutions in the materials tab)
?. [prepare in advance]
measure out 230 ml room temperature PBS for each volunteer
?. [prepare in advance]
set up the stand for leukocyte depletion using a 50 ml syringe (plunger removed), Leucoflex LXT filter and 250 ml centrifuge bottle (rinse bottles with sterile PBS, check for any cracks or warps - do not use damaged bottles)
?. [leukocyte depletion]
draw 20 ml of venous blood from a malaria infected volunteer into two 10 ml lithium heparin Vacutainers - keep at room temperature
In our hands a total of 100,000 ring-stage parasites in 20 ml whole blood (or 5,000 parasites*ml-1) will routinely generate a high quality parasite transcriptome with sufficient genome coverage to analyse expression of the subtelomeric multi-gene families (e.g. var and rifin genes in P. falciparum). See publication: https://www.medrxiv.org/content/10.1101/2020.09.04.20188144v1
?. [leukocyte depletion]
mix the blood carefully by inverting each Vacutainer ten times
?. [leukocyte depletion]
transfer the blood from both Vacutainers to one 50 ml centrifuge tube
?. [leukocyte depletion]
rinse both Vacutainers with a total of 30 ml room temperature PBS to maximise recovery of red blood cells and add to the same 50 ml centrifuge tube
?. [leukocyte depletion]
pour the 50 ml of diluted whole blood into the syringe body on your leukocyte depletion stand! take care to not create bubbles
?. [leukocyte depletion]
allow the liquid to pass through the Leucoflex LXT filter by gravity alone! do not use the plunger
?. [leukocyte depletion]
when the diluted whole blood reaches the 10 ml syringe mark (this happens quickly!) start washing the filter by adding 30 ml room temperature PBS to the syringe body! never let the filter run dry
?. [leukocyte depletion]
keep topping up the syringe until a total volume of 200 ml PBS has been used to wash the filter
?. [leukocyte depletion]
allow all liquid to pass through the filter into the 250 ml centrifuge bottle
Extensive washing maximises the recovery of red cells after leukocyte depletion. We have confirmed that infected erythrocytes and reticulocytes - even those containing mature trophozoites and schizonts - can pass through the filter.
?. [spin 1 : pellet red blood cells]
use precision scales to accurately balance all 250 ml centrifuge bottles to within 0.1 g by adding PBS drop-wise with a pasteur pipette
?. [spin 1 : pellet red blood cells]
make sure all Silicone O rings are secure and seal the bottles tightly
?. [spin 1 : pellet red blood cells]
spin at 1,000 xg for 10 min. at room temperature with the break OFF
?. [spin 1 : pellet red blood cells]
note that the Avanti centrifuge takes approx. 1 hour to slow downuse this time to:cool the centrifuge you will use for spin 2 to 4°Cinspect the Oak Ridge High-Speed Centrifuge Tubes for any cracks or warps - do not use defective tubes
?. [spin 1 : pellet red blood cells]
once the centrifuge has stopped spinning gently remove the 250 ml centrifuge bottles taking care not to disturb the delicate red cell pellet
?. [spin 1 : pellet red blood cells]
carefully aspirate and discard the supernatant with a 50 ml serological pipette using an electric pipette controller set to the lowest aspiration setting
?. [spin 1 : pellet red blood cells]
! aspirating too much supernatant will disturb the pellet and lead to red cell loss but leaving too much supernatant will reduce the concentration of saponin in the next step and impair the permeabilisation of red cell membranes
the residual volume left in the centrifuge bottle should now be approx. 15 ml (8 ml packed red cells + 7 ml supernatant)
?. [permeabilise red blood cell membranes]
resuspend the red cell pellet by gently swirling the centrifuge bottle
?. [permeabilise red blood cell membranes]
add 57 ml ice-cold working saponin solution to achieve a final concentration of 0.0075% saponin (total volume should be approx. 72 ml)
In our hands, this final working concentration of saponin will permeabilise red cell membranes but leave the parasitophorous vacuole around the malaria parasite intact. Carefully determine the optimal concentration of each batch of saponin before use (for details see buffers and solutions in the materials tab).
?. [permeabilise red blood cell membranes]
seal and invert the centrifuge bottle to mix thoroughly
?. [permeabilise red blood cell membranes]
split the saponin-red cell solution equally between two 50 ml Oak Ridge High-Speed Centrifuge Tubes and place immediately on ice! Oak Ridge tubes must not be filled above 75% capacity
?. [permeabilise red blood cell membranes]
incubate on ice for 10 min. inverting the Oak Ridge tubes every 2 min. to mix
Red cell lysis is complete when the solution becomes transparent - check by holding each tube up to the light. If necessary, extend the incubation time to 15 min.
?. [spin 2 : pellet malaria parasites]
use precision scales to accurately balance all Oak Ridge tubes to within 0.05 g by adding PBS drop-wise with a pasteur pipette
?. [spin 2 : pellet malaria parasites]
make sure all Silicone O rings are secure and seal the tubes tightly
?. [spin 2 : pellet malaria parasites]
use a permanent marker pen to indicate on each tube the expected location of the parasite pellet and place the tubes into the pre-cooled centrifuge with this mark facing outwards
?. [spin 2 : pellet malaria parasites]
If you anticipate the presence of mature parasites (such as in mixed-stage vivax infections) proceed with the next step. If you only expect to find ring-stage parasites (such as in synchronous falciparum infections) then go directly to step 37.
?. [spin 2 : pellet malaria parasites]
slow spin to pellet mature parasites : 3,000 xg for 30 min. at 4°C with the break OFFnote that the Allegra 25R centrifuge takes approx. 20 min. to slow down
?. [spin 2 : pellet malaria parasites]
gently remove the Oak Ridge tubes from the centrifuge and place onto ice
you should be able to see a small dark parasite pellet and around the pellet a smear of red cell membranes (so-called ghosts)
?. [spin 2 : pellet malaria parasites]
carefully transfer the supernatant to a new Oak Ridge High-Speed Centrifuge Tube using first a 10 ml serological pipette with an electric pipette controller set to the lowest aspiration setting and then a P1000 manual pipette! do not touch or dislodge the parasite pellet
?. [spin 2 : pellet malaria parasites]
keep the mature parasite pellet on ice
?. [spin 2 : pellet malaria parasites]
collect ring-stage parasites from the supernatant with a fast spin: 18,000 xg for 20 min. at 4°C with the break OFFnote that the Allegra 25R centrifuge takes approx. 25 min. to slow down
?. [spin 2 : pellet malaria parasites]
gently remove the Oak Ridge tubes from the centrifuge and place onto ice
you should be able to see a small dark parasite pellet and around the pellet a smear of red cell membranes (so-called ghosts)
?. [spin 2 : pellet malaria parasites]
carefully aspirate and discard the supernatant using first a 10 ml serological pipette with an electric pipette controller set to the lowest aspiration setting and then a P1000 manual pipette! do not touch or dislodge the parasite pellet
?. [wash isolated parasites to remove globin]
Washing the parasite pellet is important to remove as much globin mRNA as possible. Globin mRNA is highly abundant and very stable, and in excess will impair analysis of the parasite transcriptome.
?. [wash isolated parasites to remove globin]
If you have mature and ring-stage parasites wash these pellets separately. Remember that for each volunteer there will be two Oak Ridge tubes for each parasite fraction. Take care to choose the correct centrifugation speed.
?. [wash isolated parasites to remove globin]
add 1 ml of ice-cold PBS to the parasite pellet in the first Oak Ridge tube. rinse the walls of the tube well and mix by gently pipetting up and down at least 20 times
?. [wash isolated parasites to remove globin]
then transfer to the second Oak Ridge tube containing the same parasite fraction (mature or ring-stage) and repeat the rinse and mix step to pool together samples that were split across two tubes in step 27
?. [wash isolated parasites to remove globin]
transfer the pooled PBS-parasite solution to a 1.5 ml eppendorf tube and keep on ice
?. [wash isolated parasites to remove globin]
after spinning, gently remove the eppendorf tubes from the microfuge and place onto ice
you should be able to see a small dark parasite pellet and few red cell ghosts
?. [wash isolated parasites to remove globin]
carefully transfer the supernatant to a new 1.5 ml eppendorf tube and put the first parasite pellet back on ice
?. [wash isolated parasites to remove globin]
spin each supernatant at the appropriate speed and time for mature or ring-stage parasites (see step 46)Centrifuging the supernatant maximises parasite recovery.
?. [wash isolated parasites to remove globin]
gently remove the eppendorf tubes from the microfuge and place onto ice
you may be able to see a very small parasite pellet but very few or no red cell ghosts
?. [wash isolated parasites to remove globin]
carefully aspirate and discard the supernatant and put this second parasite pellet onto ice
?. [wash isolated parasites to remove globin]
For each volunteer and parasite fraction (mature or ring-stage) you should now have two 1.5 ml eppendorf tubes on ice, each containing a parasite pellet.
?. [preserve parasites for RNA-sequencing]
If you have mature and ring-stage parasites you can preserve them separately or pool to maximise the material available for RNA-sequencing. If choosing the second option, make sure that the volume of TRIzol used to pool all four parasite pellets does not exceed 1 ml.
?. [preserve parasites for RNA-sequencing]
for each volunteer add 1 ml TRIzol to the first parasite pellet from step 48. incubate for 5 min. in a 37°C waterbath! do not exceed this time
?. [preserve parasites for RNA-sequencing]
remove from the waterbath, wipe the tube dry and pipette up and down gently (using a P1000) until the parasite pellet has completely dissolved
?. [preserve parasites for RNA-sequencing]
transfer the TRIzol reagent to the tube containing the second parasite pellet from the same sample (step 51) and pipette gently to dissolve! do not repeat the 37°C incubation
?. [preserve parasites for RNA-sequencing]
snap-freeze each 1.5 ml eppendorf tube (containing parasites preserved in 1 ml of TRIzol) on dry ice and store at -80°C
?. [preserve parasites for RNA-sequencing]
We have used this protocol to obtain high quality/coverage P. falciparum transcriptome data (including subtelomeric var and rifin genes) from volunteers enrolled in human challenge studies. These data are publicly available from the European Nucleotide Archive (ENA) https://www.ebi.ac.uk/ena/browser/view/PRJEB33557. For details of the downstream steps required for RNA isolation, cleanup, quality control and sequencing please refer to our publication (https://www.medrxiv.org/content/10.1101/2020.09.04.20188144v1). In brief, RNA was purified using a modified phenol-chloroform extraction protocol, DNase-treated and depleted of globin and ribosomal RNA using the Globin-Zero® Gold kit (Illumina, GZG1224, now discontinued. see https://emea.illumina.com/products/selection-tools/rrna-depletion-selection-guide.html for replacements). cDNA was synthesised using a modified low-input P. falciparum Smart-Seq2 protocol (Picelli et al. 2013, PMID: 24056875, doi.org/10.1038/nmeth.2639 and Reid et al. 2018, PMID: 29580379, doi.org/10.7554/eLife.33105) and indexed libraries were generated using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (NEB E7370, an updated version of this kit is now available: NEB 7645). Libraries were sequenced on an Illumina Hiseq v4 flowcell (75 bp paired end reads).
?. [alternative downstream applications]
?. [wash isolated parasites to remove globin]
now choose the correct centrifugation speed for each tube:
?. [spin 2 : pellet malaria parasites]
keep the ring-stage parasite pellet on ice