Nucleocuvette vessel

Nucleofection of HSCs was performed as previously described^{5 (link)}. Briefly, 5.0 × 10^5 HSCs (per reaction) were resuspended in 100 μl of nucleofection buffer^{42 (link)} with 1 μg plasmid in a Nucleocuvette Vessel (Lonza). The constructs used for nucleofection: pcDNA-Flag-Yap1 (gift from Ophir Klein, Addgene plasmid #18,881), pCMV-Flag-YAP-S381A (gift from Ophir Klein, Addgene plasmid #27,377), WT AMPKα1, Kinase inactive DN mutant (gift from Kun-Liang Guan, University of California, San Diego, La Jolla, CA). Cells were nucleofected using the FF-113 program for P3 primary cells on the 4D-Nucleofector Core and X Unit system (Lonza) according to the manufacturer’s instructions. 200 μl of prewarmed media was added to the sample, which was then transferred to one well of a 12 well plate with 700 μl of prewarmed media.

The tightly synchronized mature schizont-stage parasites of P. knowlesi were transfected using the Amaxa 4D electroporator (Lonza, Basel, Switzerland) and the P3 Primary cell 4D Nucleofector X Kit L (Lonza) following previous reports (Moon et al., 2016 (link)). Briefly, a 20-μg repair template and 20 μg pCas9/sg (Mohring et al., 2019 (link)) containing sgRNA sequences for pkmsp1p were mixed with P3 Primary Cell nucleofector solution, including supplement 1 (Lonza), and transferred to a Nucleocuvette™ Vessel (Lonza), followed by nucleofection with program FP158. Transfected parasites were immediately transferred to complete media with RBCs and incubated at 550 rpm for approximately 30 min at 37°C to allow invasion before transferring to standard culture conditions. After 24 h, transfected parasites were selected by drug pressure with 100 nM pyrimethamine (Sigma-Aldrich), and the medium, including pyrimethamine, was replaced at daily intervals for 5 days. The transfected parasites were cloned out by limiting dilution and confirmed by genotyping with diagnostic primers of extracted genomic DNA (Supplementary Tables 1, 2).

REST knockdown was performed in U87 IDH-WT and IDH-MUT, the isogenic malignant glioma cell lines that genetically differed only by IDH1 mutation status. The cells were subcultured 2 days prior to the transfection so they would not exceed the confluency of 80% on the day of siRNA transfection and double transfected within 24 h, first by nucleofection, followed by lipofection. For the nucleofection, cells were trypsinized, counted, centrifuged, resuspended in Lonza SE cell line solution reagent (Lonza, PBC1-02250) and transferred to Nucleocuvette Vessel (Lonza). Control or human REST-targeting siRNA ON-TARGETplus SMARTpool (Dharmacon, D-001810–10-05 and L-006466–00-0005) was then added to the appropriate wells of the vessel and nucleofection was carried out using 4D-Nucleofector core unit. The cells were then resuspended with DMEM 10% FBS and cultured in 12 or 24 well plates (Falcon) for the next 24 h. After that time, the medium was replaced with fresh DMEM 10% FBS and the cells were transfected for the second time using Lipofectamine 2000 (Invitrogen, 2,094,065) and the same siRNA ON-TARGETplus SMARTpool (Dharmacon) as before. Protein and RNA were collected 72 h after the first transfection.