gRNAseq of RH WT T. gondii: eight passages in HFF
The libraries for next generation sequencing were generated using a 2-step PCR amplification of integrated sgRNAs. Briefly, 1000ng of genomic DNA isolated from different samples was used as template for PCR1 using forward 5 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTTCTGGTAAATGGGGATGTCAAGTT 3 and reverse 5 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGAATGACACACAGGAACTACGCG 3 primers with PCR cycling conditions of 10s 98 C, 20s 56 C, 15s 72 C for 18 cycles. PCR2 was performed using 10ng of PCR1 as template using forward 5 AATGATACGGCGACCACCGAGATCTACAC-10bp barcode- TCGTCGGCAGCGTCAGATGTG 3 and reverse 5 CAAGCAGAAGACGGCATACGAGAT-10bp barcode- GTCTCGTGGGCTCGGAGATGTGTA  3 primers with PCR cycling conditions of 10s 98 C, 20s 72 C, 20s 72 C for 16 cycles. PCR2 amplicons were uniquely dual indexed with 10 bp Illumina-compatible barcodes for each sample.