5 fluorodeoxyuridine

For each biological replicate, 400 μg of the sgRNA library were linearized with AseI, dialyzed against water, and transfected into approximately 4 × 10⁸ RH or RH/Cas9 parasites divided between 8 separate cuvettes. Transfections were used to infect 8 T-175 flasks with confluent HFF monolayers, and pyrimethamine was added 24 hours later. The parasites were allowed to egress naturally from host cells two days after infection, isolated by filtration, and 1.5 × 10⁸ parasites were passaged onto 8 T-175 flasks with fresh monolayers. The remaining parasites (~10⁷) were pelleted and stored at −80°C for analysis. This process was repeated again 5 days and 7 days post-transfection. For the drug-resistance screens, 5 μM 5-fluorodeoxyuridine (FUDR; Sigma) was added to 1.2 × 10⁷ parasites collected on day 7 post transfection, and parasites were cultured until their first lysis. Untreated mutant pools were maintained in parallel for the duration of FUDR selection. Parasite DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen) and integrated sgRNA constructs were amplified using a nested PCR with primers P74 and P75 followed by P76 and P77. The resulting libraries were sequenced on a HiSeq 2500 (Illumina) with single-end reads using primers P150 and P151.

Bovine adrenal glands (from male animals) were obtained from a local slaughterhouse and chromaffin cells were isolated by digestion of the adrenal medullae with collagenase followed by density gradient centrifugation (Todd et al. 2012 (link)). For patch clamp experiments cells were plated onto collagen coated coverslips in 35mm tissue culture dishes at a density of ~ 0.1 – 0.15 x 10⁶ cell / mL. For catecholamine secretion experiments the cells were plated in 24-well tissue culture plates at a density of ~ 0.3 x 10⁶ cells per well. Cells were maintained in a humidified incubator at 37 °C and 5% CO₂ in culture medium that consisted of Dulbecco’s modified Eagle medium \ F12 (1:1) supplemented with 10 % fetal bovine serum, 2mM glutamine, penicillin/streptomycin (100 unit mL⁻¹/100 μg mL⁻¹), 10 μM cytosine arabinoside (Sigma-Aldrich; St Louis MO) and 10 μM 5-fluorodeoxyuridine. All tissue culture reagents were from Life Technologies (Grand Island, NY) unless noted otherwise. Fibroblast proliferation was suppressed with cytosine arabinoside leaving relatively pure chromaffin cell cultures. The culture medium was replaced the day after isolation and experiments were performed 2–5 days following cell isolation.