TbRAP1's dsDNA binding activity plays an important role in subtelomeric gene expression regulation

We examine the function of the TbRAP1 DB domain in gene expression regulation in Trypanosoma brucei that causes human African trypanosomiasis. TbRAP1 is required for normal VSG monoallelic expression, a key aspect of antigenic variation that is used by T. brucei to evade the host immune response. Overall design: High-throughput paired-end RNA sequencing on the Illumina platform was performed. Mutant T. brucei strain has one WT TbRAP1 allele flanked by loxP repeats (floxed) and a FLAG-HA-HA and an SV40 NLS- tagged TbRAP1-5A, TbRAP1-2SD, or a deleted allele. Control T. brucei strain has one floxed and a WT TbRAP1 allele. Both mutant and control cells were induced for Cre induction for 30 hrs (to remove the floxed TbRAP1 allele) before total RNA was isolated. Three independent induction were performed for both cells. RNA samples were sent to Novogene for subsequent experiments. Poly(A) RNA was purified for generating cDNA library and paired-end sequencing was done using Illumina. Gene expression profiles were compared between the mutant and control cells by differential gene expression analysis.