Total parasite RNA extraction.

Materials

• Eliminase or RNase Zap
• TRIzol LS (Ambion #10296010)
• Safelock tubes (Eppendorf #022600044)
• Qiagen stainless steel 5 mm beads (#69989)
• Nuclease-free water
• Autoclaved MilliQ water
• Zymo Direct-zol RNA Miniprep (#R2051) — for option A
• 100% molecular grade ethanol — for option A
• Chloroform — for option B
• Isopropanol — for option B

Tissue storage

1. Treat the working area with Eliminase or RNase Zap, acquire nuclease-free filtered tips, and procure liquid nitrogen or dry ice.

2. Update the Parasite Tissue and Nucleic Acid Inventory with the tissues being prepared for storage, and use cryolabels to label 1.5 mL tubes with the Sample ID. Include a lid label with stage and T###.

3. Prepare the tissue samples according to stage:

   Stage	Wash method	Ideal quantity
   mf	Use RPMI-1640 and a PD-10 column to filter the mf. Wash once with nuclease-free water (MilliQ).	1–3 million
   L3	Wash L3 in a 15 mL conical tube using a small volume of nuclease-free water (MilliQ). Allow L3s to pellet by gravity.	300+
   Adult	Wash the adult parasite cluster in a Petri dish containing nuclease-free water (MilliQ).	30–50 ¹

   ¹ Per the TRIzol LS protocol, 50–100 mg tissue is recommended per 750 μL reagent.

4. Transfer washed parasites to a nuclease-free 1.5 mL microcentrifuge tube, pellet the parasites, and remove as much supernatant as possible — 100 μL max residual.

5. Add 300 μL TRIzol LS.

6. Snap freeze in liquid nitrogen or dry ice.

7. Store at -80°C in the parasite tissue storage box.

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   If time allows, perform the RNA extraction immediately.

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