Trizol was added to bradyzoites for the preservation of RNA, and the RNA extracted using phenol/chloroform separation and isopropanol precipitation. Samples include brain enriched bradyzoites at 28 DPI (n = 2), 90 DPI (n = 2), and 120 DPI (n = 2). Ten nanogram of total RNA was processed using the SMART-Seq v4 Ultra Low Input RNA Kit (Takara), using 7 rounds of cDNA amplification. One nanogram of cDNA was indexed using the Nextera XT Index Kit (Illumina). RNA libraries were multiplexed and run across 2 lanes for paired end, 125 base pair sequencing by Illumina HiSeq 2500 at the University of Wisconsin Biotechnology Center.
Tachyzoite RNA was processed from the ME49 parasites grown in human foreskin fibroblasts (HFF) cells. Just before lysis of the parasite vacuole, infected HFF cells were scraped in trizol and RNA extracted as stated above. RNA libraries were prepared using a TruSeq RNA Library Prep kit v2 set A (Illumina) and sequenced using paired-end sequencing as stated above.
Tachyzoite RNA was processed from the ME49 parasites grown in human foreskin fibroblasts (HFF) cells. Just before lysis of the parasite vacuole, infected HFF cells were scraped in trizol and RNA extracted as stated above. RNA libraries were prepared using a TruSeq RNA Library Prep kit v2 set A (Illumina) and sequenced using paired-end sequencing as stated above.