ABSTRACT: ZC3H5-TAP expressing cells were transfected with RNAi targeting ZC3H5. Two clones where used and samples collected 9 and 12 hours after RNAi induction (Tetracycline 250ng/ml).
Project description:Gradient fractions of RNAi of XAC1 (Tb927.7.2780) in Trypanosoma brucei bloodstream forms. RNAi was induced using tetracycline and cell extracts were fractionated into polysomal and monosome-non-ribosome-associated fractions.
Project description:RBSR2-TAP expressing cells were transfected with RNAi targeting RBSR2. 3 independent clones were used and RNAi was induced for 24 hours with 100ng/ml tetracycline. Cell lines expressing RBSR2-CTAP with no RNAi construct were used as controls. These are actually paired-end reads, file names ending _1 are one strand and _2 are the other strand.
Project description:RBSR1-CTAP expressing cells were transfected with RNAi targeting RBSR1. 3 independent clones were used and RNAi was induced for 24 hours with 100ng/ml tetracycline. PolyA enriched samples are paired-end reads, file names ending _1 are one strand and _2 are the other strand. rRNA depleted sample are single end reads.
Project description:Bloodstream-form trypanosomes (Lister 427) constitutively expressing ZC3H5-TAP (Tb927.3.740) were used. The protein was pulled down with an IgG column , then the protein and bound RNA was eluted using TEV protease. RNA was then sequenced from unbound (flow-through) and bound (eluate) fractions.
Project description:A procyclic form Trypansome brucei RNAi line (PTT parental line, transfected with pALC14 incorporating a TbNMD3 gene fragment) capable of inducing depletion of TbNMD3 was analysed for mRNA expression by RNAseq Cells were grown for 72 hours in culture; RNAi was induced in cells by the addition of 1 microgram/ml of tetracycline
Project description:ZC3H5 was knocked down in bloodstream form T.brucei by RNAi for 0h and 12h. WT bloodstream form cells were used as control. Polysome fractionation was performed, RNA was purified from each fraction and fractions were pooled the following: F=free fraction; M=monosomes; L=light polysomes; H=heavy polysomes. RNA was depleted and RNA-Seq was performed.
Project description:Trypanosomes have seven cullin paralogs, the majority lineage-specific. Six of those cullins (TbCul-A, TbCul-C, TbCul-D TbCul-E, TbCul-F and TbCul-G) were endogenously 3xHA-epitope tagged at the C-terminus. We used affinity capture/mass spectrometry to determine the composition of these cullin complexes. TbCul-A and TbCul-E function were further studied by analysis of proteome changes upon upon RNAi.
Project description:As an additional strategy for investigating T. brucei transcriptome responsiveness, the mRNA of a highly important protein, the variant surface glycoprotein (VSG) the major surface protein in the bloodstream stage, was suppressed with RNAi. VSG RNAi results in arrest of cell cycle progression in the bloodstream stage. In addition, the mRNA of a highly important protein for endocytosis, the clathrin heavy chain (CLH), was suppressed with RNAi. CLH knockdown leads to a complete block to endocytosis. We hypothesized that if trypanosomes were able to sense alterations in trafficking and respond to these changes, then depletion of these ORFs by RNAi would be expected to elicit a response at the transcriptome level.<br> <br> part 1: 2 biological replicates of SMB cells transfected with the VSG- RNAi vector grown under normal conditions (non-induced), and 2 replicates of the same cells treated with 1 ug/ml tetracycline (induced) for 24hr, as well as dye swaps were used.<br> <br> part 2: 2 biological replicates of SMB cells transfected with the VSG- RNAi vector grown under normal conditions (non-induced), and 2 replicates of the same cells treated with 1 ug/ml tetracycline (induced) for 3 days, as well as dye swaps were used.<br> <br> part 3: 3 biological replicates of SMB cells transfected with the CLH- RNAi vector grown under normal conditions (non-induced), and 3 replicates of the same cells treated with 1 ug/ml tetracycline (induced), as well as dye swaps were used.