Taq based master mix

For tissue-level analysis, one-fourth of a mouse brain was placed in 1 mL Trizol (Ambion), mechanically homogenized using 1 mm zirconia/silica beads (Biospec) for 30 seconds using a Mini-BeadBeater 16 (BioSpec). For gene expression analysis of magnetically-enriched cells, cells were homogenized in Trizol by pipetting. RNA was extracted from Trizol according to manufacturer’s instructions (Invitrogen). High Capacity Reverse Transcription Kit (Applied Biosystems) was used to generate cDNA. Quantitative PCR was performed using 2X Taq-based Master Mix (Bioline) and TaqMan gene expression assays (Applied Biosystems), or custom primers (Integrated DNA Technologies), run on a CFX384 Real-Time System thermocycler (Bio-Rad Laboratories). Murine Hprt and T. gondii Act1 were used for normalization for analyzing host and parasite gene expression, respectively, and relative expression is reported as 2^(−ΔΔCT). The following Thermo Fisher mouse gene probes were used: Stat1 (Mm00439518_m1), Hprt (Mm00446968_m1), Ifng (Mm01168134_m1), Nos2 (Mm00440502_m1), Il6 (Mm00446190_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), Tnfa (Mm00443258_m1), Ccl2 (Mm00441242_m1), Ccl5 (Mm01302427_m1), Cxcl9 (Mm00434946_m1), Cxcl10 (Mm00445235_m1). custom primers for used for analyzing T. gondii genomic DNA and gene expression were used and are provided in S1 Table.

Approximately ¼ of a mouse brain was put into 1 mL TRIzol (Ambion) in bead-beating tubes (Sarstedt) containing 1 mm zirconia/silica beads (BioSpec). Tissue was homogenized for 30 s using a Mini-bead beater (BioSpec). RNA was extracted according to the manufacturer’s instructions (Ambion). High Capacity Reverse Transcription Kit (Applied Biosystems) was used for cDNA synthesis. qRT-PCR was done using 2X Taq-based Master Mix (Bioline) and TaqMan gene expression assays (Applied Biosystems). Reactions were run on a CFX384 Real-Time System (Bio-Rad Laboratories). HPRT was used as the housekeeping gene for all reactions and relative expression was calculated as 2^(−ΔΔCT). Primer assays were acquired from ThermoFisher and include Vcam1 (Mm01320970_m1), Icam1 (Mm00516023_m1), Ccl2 (Mm00441242_m1), and Il1a (Mm00439620_m1).